BRPI0608455A2 - a composition comprising cat allergen conjugates as well as vaccines, pharmaceutical compositions and medicaments containing them, and a process for their production - Google Patents

Abstract

COMPOSITION UNDERSTANDING CAT ALLERGEN ASSEMBLIES AS WELL AS VACCINES, PHARMACEUTICAL COMPOSITIONS, AND MEDICINES CONTAINING THE SAME, AND PROCESS FOR THEIR PRODUCTION. The present invention relates to the fields of medicine, public health, immunology, molecular biology and virology. The invention provides compositions comprising a virus-like particle (VLP) or a viral particle and at least one antigen, particularly at least one feline antigen, and more particularly at least one feline antigen which is a human allergen. In certain embodiments, the antigen is a Fel dl antigen, or fragment thereof, covalently linked to the VLP. The invention also provides processes for producing the compositions. The compositions of the invention induce efficient immune responses, particularly antibody responses, in mammals, particularly humans. The compositions and processes of the invention are useful in the production of vaccines, particularly for the treatment and / or prevention of allergies to cat dander and other cat antigens and allergens.

Description

Report of the Invention Patent for "Cat Allergen Conjugates and Uses of the Same".

Background of the Invention Field of the Invention

The present invention relates to the fields of medicine, health

immunology, molecular biology and virology. The invention provides compositions comprising a virus-like particle (VLP) or a virus particle and at least one antigen, particularly at least one feline antigen, and more particularly at least one feline antigen which is a human allergen. In certain embodiments, the antigen is a Fel d1 antigen, or a fragment thereof, covalently linked to the VLP. The invention also provides processes for producing the compositions. The compositions of the invention induce efficient immune responses, in particular antibody responses, in mammals, particularly humans. The compositions and processes of the invention are useful in the production of vaccines, in particular for the treatment and / or prevention of cat coat allergies and other cat antigens and allergens. Related Technique

The domestic cat (Felis domesticus) is an important source of internal allergens (Lau, S. et al. (2000) Lancet 356, 1392-1397). In fact, cats are found in approximately 25% of households in western countries and cat allergy is found in a large part of the population. Symptom severity ranges from mild rhinitis and conjunctivitis to life-threatening exacerbated asthma.

Although patients are occasionally sensitized by several different molecules present in dandruff and cat hair, the main allergen is Fel d 1 (ie, Felis domesticus allergen 1, formerly Cat 1, ie Cat 1 allergen). The importance of this allergen has been highlighted in numerous studies. In fact, more than 80% of cat allergic patients exhibit IgE antibodies against this potent allergen (van Ree, R. et al. (1999) J. Al-lergyClin ImmunoM 04, 1223-1230).

Fel d1 is a 35-39 kDa acid glycoprotein containing 10-20% N-linked carbohydrates and is present in cat hair, saliva and tear glands. It is formed by two non-covalently bonded heterodimers. Each heterodimer is comprised of a 70-residue peptide (known as "chain 1") and a 78, 85, 90, or 92-residue peptide (known as "chain 2"), which are encoded by different genes (see Duffort, (1991) Mol Immunol 28, 301-309; Morgenstern, JP et al., (1991) Proc Natl Acad Sci USA 88, 9690-9694 and Grif-fith, IJ (1992) Gene 113 , 263-268).

Treatment of cat allergic patients is currently performed by desensitizing therapy involving repeated injections with increasing doses of a crude cat dander extract or Fel d1-derived short peptides. Lilja et al and Hedlin et al described a desensitization scheme during which crude cat dandruff extracts were provided to cat allergic patients (Lilja, Q et al. (1989). J Allergy Clin Immunol 83, 37-44 and Hedlin et (1991) J Allergy Clin Immunol 87, 955-964). This regimen took at least two to three years and patients after three years of treatment still had systemic symptoms. The use of short peptides derived from Fel d1 for desensitization resulted in no significant differences between the peptide group and the placebo group (Oldfield, W. L. et al. (2002) Lancet 360, 47-53). Efficacy was only observed when a large amount (750 æg) of the short peptide was provided to patients (Norman, P. S. et al. (1996) Am J Respir Crit Care Med 154, 1623-1628).

Allergic side effects such as late asthmatic reactions have been reported in both treatment with crude cat dandruff extract and treatment with short peptide. Therefore, anaphylactic shock from injected allergen is an important safety issue in any desensitization scheme. However, preventing this effect by reducing the amount of allergen injected either reduces the effectiveness of the treatment or prolongs the treatment. Thus, there is a great need in the area of cat allergy treatment for alternative desensitization regimens that are capable of reducing allergic symptoms but not triggering the side allergic reaction. Summary of the Invention

It has now surprisingly been found that compositions and vaccines of the invention comprising, respectively, at least one Fel d1 antigen, or fragment thereof of the invention, are not only capable of inducing immunological responses against Fel d1, and hence in the present invention. particular antibody responses, capable of desensitizing cat allergy patients and, in particular, within a short period of time, indicating the high efficacy of the compositions and vaccines of the invention, respectively. Furthermore, it was surprisingly found that the Fel d1 of the invention, when covalently linked to VLP according to the invention, dramatically reduced anaphylactic activity when compared to the Fel d1 of the invention which was covalently linked to VLP, while maintaining a high degree. antigenicity and immunogenicity. This represents a major advantage over prior art cat allergy treatments since the inventive compositions and vaccines, respectively, dramatically reduce the risk of causing anaphylactic shock in animals and humans to be immunized. In addition, the compositions and vaccines of the invention, respectively, allow the antigen to be delivered at much higher doses compared to prior art cat allergy treatments, which in turn improves the efficacy and / or shortens the duration of the antigen. total desensitization scheme. Thus, the compositions and vaccines of the invention, respectively, induce potent immune responses against Fel d1, but without triggering an allergic reaction.

Thus, in the first feature, the present invention provides a composition comprising (a) a central particle with at least one attachment site, wherein said central particle is a virus-like particle (VLP) or a viral particle, and (b) at least one antigen with at least a second attachment site, wherein said at least one antigen is a Fel d1 protein, or fragment of Fel d1, and wherein (a) and (b) are covalently linked. by at least one said first attachment site and at least one said second attachment site, preferably to form an ordered and repeated antigen array.

In another feature, the present invention provides a vaccine composition. In addition, the present invention provides a process for administering the vaccine composition to a human or non-human mammal such as a cat allergic dog, preferably cat Fel d1. In a preferred embodiment, the vaccine composition further comprises at least one adjuvant. The vaccine composition of the invention is, however, capable of inducing a strong immune response, particularly antibody response, without the presence of at least one adjuvant. Thus, in one preferred embodiment, the vaccine is devoid of an adjuvant. Prevention of adjuvant use may reduce the possible occurrence of side effects related to adjuvant use.

In a preferred embodiment, the VLP included in the composition and vaccine composition, respectively, is produced by recombination into a host and the VLP is essentially free of host RNA, preferably free of host nucleic acids. It is advantageous to reduce, or preferably eliminate, the amount of host, preferably free of host nucleic acids, to prevent unwanted T cell responses, as well as other unwanted side effects such as fever.

In a preferred embodiment, the composition of the invention further comprises at least one immunostimulatory substance, preferably at least one immunostimulatory nucleic acid. In yet another preferred embodiment, the immunostimulatory nucleic acid is contained in the VLP of the invention. Inclusion of immunostimulatory substances, preferably immunostimulatory nucleic acids, in the composition of the invention may direct immune responses to Th1 responses and thereby suppress Th2 responses and thereby suppress IgE production. In one aspect, the present The invention provides a method for treating cat allergy by administering the composition or vaccine of the invention, respectively, to a cat allergic target, preferably human.

In yet another aspect, the present invention provides a pharmaceutical composition comprising the composition of the invention and an acceptable pharmaceutical carrier.

In another aspect, the present invention provides a process for producing the composition of the invention comprising (a) a central particle with at least one attachment site, wherein said central particle is a virus-like particle (VLP) or a particle. viral; (b) providing at least one antigen with at least a second attachment site, wherein said antigen is a Fel d1 protein or Fel d1 fragment; and (c) combining said central particle and said at least one antigen to produce said composition, wherein said at least one antigen and said central particle are bonded by at least said first and at least one antigen. , said second attachment sites.

In one aspect, the invention provides a Fel d1 fusion protein, comprising Fel d1 chain 1 and Fel d1 chain 2, fused by an amino acid spacer, which connects the N-terminus of a chain to C- terminal of the other chain, wherein said amino acid spacer is an amino acid sequence of 10-30 amino acid residues and wherein said fusion protein is produced in E.coli or wherein said fusion protein is not It is glycosylated. Brief Description of the Figures

FIG 1 shows the purified renatured and Fel d1 fusion proteins stained by Coumassie on a non-reducing SDS-PAGE gel. The samples in lane 1 used DTT as reducing agent. The samples in lane 2 did not use DTT.

FIG 2 shows the degranulation of basophils by isolated Fel d1 fusion proteins or by Fel d1 fusion proteins coupled to Qp. The X axis represents the concentration of the corresponding Fed d1 protein. The Y axis represents the percentage of basophils that have been deflated.

Figure 3 shows the results of the prick test of a cat allergic volunteer who received Qp-FELDI on days 0, 7 and 14 and the tests were also conducted on days 0, 14 and 21.

FIG 4A shows the nasal dose escalation score and overall nasal score (FIG 4B) of a cat allergic volunteer who received Qp-FELD 1 at said 0, 7 and 14 and the tests were conducted on day 0, 14e21.

Detailed Description of the Invention

Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those of ordinary skill in the art to which this invention belongs.

Adjuvant: The term "adjuvant" as used herein refers to non-specific immune response enhancers or substances that enable the generation of a host accumulation which, when combined with the vaccine and pharmaceutical composition, respectively, of the present invention may provide an even stronger immune response. A variety of adjuvants may be used. Examples include complete and incomplete Freund's adjuvant, aluminum hydroxide and modified muramyldipeptide. Other adjuvants are mineral gels such as aluminum hydroxide, surface active substances such as lysoleicithin, pluronic polyols, polyanions, peptides, oily emulsions, keyhole limpet hemocyanin, dinitrophenol and potentially useful human adjuvants such as BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum. . These adjuvants are also well known in the art. Other adjuvants that may be administered with the compositions of the invention include, but are not limited to, monophosphoryl lipid immunostimulants, AdjuVax 100a, QS-21, QS-18, CRL1005, aluminum salts (Alum), MF-59, OM-174, OM-197, OM-294 and virosomal technology adjuvants. Adjuvants may also comprise a mixture of these substances. VLP has been generally described as an adjuvant. However, the term "adjuvant" as used in the context of this application refers to adjuvants other than the VLP being used for the compositions of the invention, in addition to said VLP.

Antigen: As used herein, the term "antigen" refers to a molecule capable of binding to an antibody or a T cell receptor (TCR), if presented by MHC molecules. The term "antigen" as used herein also encompasses T-cell epitopes. Umantigen is also capable of being recognized by the immune system and / or inducing a humoral immune response and / or cellular immune response that leads to lymphocyte activation. B and / or T. This may, however, require that, at least in certain cases, the antigen contains or is linked to a Th cell epitope and is provided in adjuvant. An antigen may have one or more epitopes (epitopes B and T). The specific reaction, referred to above, is intended to indicate that the antigen will preferably react, typically highly selectively, with its corresponding antibody or TCR and not with the multiplicity of other antibodies or TCRs that may be incited by other antigens. Antigens, as used herein, may be mixtures of several individualized antigens.

Antigenic Site: The term "antigenic site" and the term "antigenic epitope", which are used interchangeably herein, refer to continuous or discontinuous portions of a polypeptide, which may bind to an antibody or a T cell receptor, in the context of an MHC molecule. Immunospecific binding excludes non-specific binding, but does not necessarily exclude cross-reactivity. The antigenic site typically comprises 5-10 amino acids in a special conformation exclusive to the antigenic site.

Associated: The term "associated" (or its noun, association) as used herein refers to all possible ways, preferably by chemical interactions, by which two molecules are joined. Chemical interactions include covalent and noncovalent interactions. Typical examples of non-covalent interactions are ionic interactions, hydrophobic interactions or hydrogen bonds, while covalent interactions are based, by way of example, on covalent bonds such as ester, ether, phosphate ester, amide, peptide, carbon-phosphorus and carbon bonds. sulfur, such as thioether or imide bonds.

First site: As used herein, the term "first site" refers to an element that occurs naturally with VLP or is artificially added to the VLP, and to which the second site may be attached. . The first attachment site may be a protein, amino acid, peptide, sugar, polynucleotide, natural or synthetic polymer, metabolite or secondary compound (biotin, fluorescein, retinol, digoxigenin, metal ions, phenylmethyl sulfonyl fluoride) or chemically reactive group such as an amino group, a carboxyl group, a sulfhydryl group, a hydroxyl group, a guanidine group, a histidine group or a combination thereof. A preferred embodiment of the first attachment site being a chemically reactive group is the amino group of an amino acid such as lysine. The first attachment site is typically located on the surface and preferably on the outer surface of the VLP. There are several first surface attachment sites, preferably on the outer surface, of the seed to virus particle in a repeated configuration. In a preferred embodiment, the first attachment site is associated with the VLP by at least one covalent bond, preferably by at least one peptide bond. In another embodiment, the first attachment site occurs naturally with the VLP.

Alternatively, in one preferred embodiment, the first attachment site is artificially added to the VLP.

Second Fixation Site: As used herein, the term "second fixation site" refers to a naturally occurring or artificially added element to the Fel d1 of the invention to which the first fixation site may attach. The second Fel d1 attachment site of the invention may be a protein, polypeptide, amino acid, sugar, polynucleotide, natural or synthetic polymer, metabolite or secondary compound (biotin, fluorescein, retinol, digoxigenin, metal ions). , phenylmethyl sulfonyl fluoride) or a chemically reactive group such as an amino group, a carboxyl group, a sulfhydryl group, a hydroxyl group, a guanidine group, a hitidine group or a combination thereof. A preferred embodiment of a second attachment site being a chemically reactive group is the sulfhydryl group, preferably of a cysteine amino acid. The terms "Fel d1 of the invention with at least one second attachment site" therefore refer to a construct comprising the Fel d1 of the invention and at least a second attachment site. However, particularly for a second, non-naturally occurring second attachment site in the Fel d1 of the invention, this construct still typically and preferably comprises a "connector". In another preferred embodiment, the second attachment site is associated with the Fel d1 of the invention by at least one covalent bond, preferably by at least one peptide bond. In yet another embodiment, the second attachment site occurs naturally in Fel d1 of the invention. In yet another preferred embodiment, the second attachment site is artificially added to the Fel d1 of the invention by means of a linker, wherein said linker comprises or is alternatively constituted by a cysteine. Preferably, the linker is fused to the Fel d1 of the invention by a peptide bond.

Coating protein: The term "coat protein" and the term "capsid protein", used interchangeably in this application, refers to a viral protein, preferably a subunit of a natural virus capsid, preferably phage RNA. , which is capable of being incorporated into a viral capsid or VLP.

Fel d1 of the invention: The term "Fel d1 of the invention" as used herein refers to at least one Fel d1 protein or at least one Fel d1 fragment.

Fel d1 Chain 1: The term "Fel d1 Chain 1" as used herein refers to a polypeptide comprising or alternatively consisting of an amino acid sequence such as SEQ IDNO: 22 or a homologous sequence thereof . The term "homologous sequence of SEQ ID NO: 22" as used herein refers to a polypeptide having an identification with respect to SEQ ID NO: 22 greater than 70%, preferably greater than 80%. %, more preferably greater than 90%, and even more preferably greater than 95%. The term "Fel d1 chain 1" as used herein shall also refer to a po-lipeptide comprising at least one post-translational modification, including, but not limited to, at least one glycosylation, of the d 1 chain. Fel d1 as defined herein. Preferably, the Fel 1 chain 1 as defined herein is comprised of a maximum of 130, even more preferably of a maximum of 100 amino acids in total.

Fel d1 Chain 2: The term "Fel d1 Chain 2" as used herein refers to a polypeptide comprising or alternatively consisting of an amino acid sequence such as SEQ ID NO: 23, SEQ ID NO : 25 or SEQ ID NO: 26, or a homologous sequence thereof. The term "homologous sequence of SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 26" as used herein refers to a polypeptide having an identification with respect to SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 26, greater than 70%, preferably greater than 80%, more preferably greater than 90%, and even more preferably greater than 95%. The term "Fel d1 chain 2" as used herein shall also refer to a polypeptide that encompasses at least one posttranslational modification, including, but not limited to, at least one Fel d1 chain 2 glycosylation, as used herein. Preferably, the Fel d1 chain 2 as defined herein is comprised of a maximum of 150, more preferably of a maximum of 130, even more preferably of a maximum of 100 amino acids in total.

Fel d1 protein: The term "Fel d1 protein" as used herein refers to a protein that comprises or alternatively comprises Fel d1 chain 1 and Fel d1 chain 2. Preferably, Fel d1 chain 1 and Fel d1 chain 2 are covalently bonded. In one embodiment, Fel d1 chain 1 and Fel d1 chain 2 are linked via at least one disulfide bond. In another preferred embodiment, chain 1 and chain 2 are fused directly or by means of a spacer, in which case said Fel d1 protein further comprises or consists of a spacer. Preferably, the Fel d1 protein as defined herein is comprised of at most 300, even more preferably at most 200 amino acids in total. Typically and preferably, the Fel d1 protein according to the invention is capable of inducing in vivo the production of antibodies that specifically bind to the natural Fel d1 protein or recombinant Fel d1 as produced according to EXAMPLE 5 of the present invention.

Fel d1 Fragment: The term "Fel d1 fragment" as used herein refers to a polypeptide comprising or alternatively comprising at least one Fel d1 antigenic site. Typically and preferably, the term "Fel dl fragment" as used herein refers to a polypeptide comprising or alternatively consisting of at least two Fel d1 antigenic sites. Preferably, the antigenic sites are covalently linked, more preferably, the antigenic sites are linked by at least one peptide bond, and in which case a spacer may be required between the antigenic sites. Preferably, the at least two antigenic sites are derived from Fel d1 chain 1 and Fel d1 chain 2. Preferably, the Fel d1 fragment as defined herein is comprised of a maximum of 130, more preferably of a maximum of 100 and even more preferably of a total of 60 amino acids. Typically and preferably, the Fel d1 fragment is capable of inducing in vivo the production of antibodies that specifically bind to the natural Fel d1 protein or recombinant Fel d1 as produced according to EXAMPLE 5 of the present invention.

Bound: The term "bound" (or its noun: bond) as used herein refers to all possible ways, preferably chemical interactions, whereby at least one first attachment site and at least the second site Fasteners are joined. Chemical interactions include covalent and noncovalent interactions. Typical examples for non-covalent interactions are tonic interactions, hydrophobic interactions or hydrogen bonds, while covalent interactions are based, by way of example, on covalent bonds such as ester, ether, esterphosphate, amide, peptide, carbon-phosphorus, and carbon-carbon bonds. sulfur as thioether or imide bonds. In certain embodiments of preference, the first attachment site and the second attachment site are linked via a covalent bond, preferably through at least one non-peptide bond, and most preferably via exclusively non-peptide bond. peptides. The term "bound" as used herein, however, will encompass not only a direct attachment of at least the first attachment site and at least the second attachment site, but also, alternatively and preferably, an indirect attachment. at least the first fixation site and at least the second fixation site by intermediate molecule (s) and, typically, preferably presently using at least one heterobifunctional crosslinker.

Connector: A "connector" as used herein associates the second attachment site with the Fel d1 of the invention or already comprises, consists essentially of or comprises the second attachment site. Preferably, a "linker" as used herein already comprises the second attachment site, typically and preferably - but not necessarily - in the form of an amino acid residue, preferably a cysteine residue. A "linker" as used herein is also referred to as "amino acid linker", particularly when a linker according to the invention contains at least one amino acid residue. Accordingly, the terms "linker" and "amino acid linker" are used interchangeably herein. However, this does not imply that a linker consists exclusively of amino acid residues, although a linker consisting of amino acid residues is a preferred embodiment of the present invention. Amino acid residues are preferably composed of naturally occurring amino acids or unnatural amino acids known in the art, all L or all D or mixtures thereof. Preferred additional embodiments of a linker according to this invention are molecules comprising a sulfhydryl group or a cysteine residue and these molecules are, therefore, also encompassed by this invention. Other linkers useful for the present invention are molecules comprising a C1-C6 alkyl moiety, a cycloalkyl such as a cyclopentyl or cyclohexyl, aryl or heteroaryl moiety. In addition, linkers preferably comprising a C1-C6 alkyl, C5, C6 cycloalkyl, aryl or heteroaryl moiety and additional amino acid (s) may also be used as linkers for the present invention and will be within the scope of the invention. . The association of the linker with the Fel d1 of the present invention is preferably by at least one covalent bond, more preferably by at least one peptide bond.

Ordered and repeated antigen array: As used herein, the term "ordered and repeated antigen array" generally refers to an antigen pattern that is repeated or categorized, typically and preferably, by a high degree of uniformity in the spatial arrangement of antigens. relative to the virus-like particle, respectively. In one embodiment of the invention, the repeated pattern may be a geometric pattern. Certain embodiments of the invention, such as phage RNA-coupled antigens, are typical and preferred examples of suitable ordered and repeated antigenic arrangements, which, moreover, have rigorously repeated paracrystalline orders of antigens, preferably with 1 to 30 nanometer spaces, preferably from 2 to 15 nanometers, even more preferably from 2 to 10 nanometers, and once again even more preferably from 2 to 8 nanometers and even more preferably from 1.6 to 7 nanometers.

Packaged: The term "packaged" as used herein refers to the state of a polyanionic macromolecule or immunostimulating substances with respect to VLP. The term "packaged" as used herein includes bonding which may be covalent, for example by chemical coupling, or non-covalent, for example, ionic interactions, hydrophobic interactions, hydrogen bonds, and the like. The term also includes the confinement, or partial confinement, of a polyanionic macromolecule. Thus, the polyanionic macromolecule or immunostimulatory substances can be confined by VLP without the existence of a bond, particularly a covalent bond. In preferably embodiments, the at least single polyanionic macromolecule or immunostimulants are packaged within the VLP, most preferably non-covalently.

Spacer: The term "spacer" as well as its equally used term "amino acid spacer" as used herein refers to an amino acid sequence segment that is no longer than 30 amino acids and which connects the N-terminal of a chain with the C-terminal of another chain of Fel d1.

Viral Particle: The term "viral particle" as used herein refers to the morphological form of a virus. In some types of viruses, it comprises a genome surrounded by a protein capsid; others have additional structures (eg envelopes, tails, etc.).

Virus-like particle (VLP) as used herein refers to a non-replicating or non-infectious virus, preferably a non-replicating non-infectious viral particle, or refers to a particle resembling a non-replicating virus. and non-infectious, preferably a structure resembling a non-replicating non-infectious virus, preferably a virus capsid. The term "non-replicating" as used herein refers to being unable to replicate the genome included in the VLP. The term "non-infectious" as used herein refers to being unable to enter the host cell. Preferably, a virus-like particle according to the invention is non-replicating and / or non-infectious since it lacks all or part of the viral genome or genomic function. In one embodiment, a virus-like particle is a viral particle in which the viral genome has been physically or chemically inactivated. Typically and most preferably, a virus-like particle does not have all or part of the infectious replicating elements of the viral genome. A virus-like particle according to the invention may contain nucleic acid distinct from its genome. A typical and preferred embodiment of a virus-like particle according to the present invention is a viral capsid, such as the corresponding viral virus capsid, bacteriophage, preferably phage RNA. The term "viral capsid" or "capsid" refers to the macromolecular assembly composed of viral protein subunits. Typically, there are 60,120, 180, 240, 300, 360 and over 360 viral protein subunits. Typically and preferably, interactions of these subunits lead to the formation of viral capsid or viral capsid-like structure with an inherent repetitive organization, wherein said structure is typically spherical or tubular. For example, phage or HBcAgs RNA capsids have a spherical form of icosahedral symmetry. The term "capsid-like structure" as used herein refers to a macromolecular assembly composed of similar viral protein subunits. to capsid morphology in the sense defined above, but deviates from the typical symmetrical assembly while maintaining a sufficient degree of order and repetition.

A common feature of viral particle and virus-like particle is the highly ordered and repetitive arrangement of its subunits.

Virus-like particle of a phage RNA: As used herein, the term "virus-like particle of a phage RNA" refers to a virus-like particle that comprises or preferably consists essentially of or consists of proteins. coatings, mutants or fragments thereof, of a phage RNA. In addition, a virus-like particle of a phage RNA resembles the structure of a phage RNA, being non-replicating and / or non-infectious and devoid of at least the gene or genes encoding the phage RNA replication mechanism and, typically, it is also devoid of the gene or genes encoding the protein or proteins responsible for attachment or entry into the host. This definition should, however, also encompass phage RNA virus-like particles, where the above-mentioned gene or genes are still present but are inactive, and thus also leading to virus-like particles of a non-replicating phage RNA. and / or not infectious. Preferably VLPs, derived from phage RNA, exhibit icosahedral symmetry and consist of 180 subunits. In this disclosure, the term "subunit" and "monomer" are used interchangeably and equally within this context. In this application, the term "phage RNA" and the term "bacteriophage RNA" are used interchangeably. Preferably used methods for producing a virus-like particle of a non-replicating and / or non-infectious phage RNA is by physical and chemical inactivation such as UV radiation, formaldehyde treatment and typically and preferably by genetic manipulation.

One or one: When the terms "one" or "one" are used in this disclosure, they mean "at least one" or "one or more" unless otherwise indicated.

Identification of the amino acid sequence can be terminated by the traditional process using known computer programs such as the Bestfit program. When using the Bestfit or any other sequence alignment program, preferably using Bestfit, to determine if a specific sequence is, for example, 95% identical to a reference amino acid sequence, the parameters are set such that The identification percentage is calculated over the entire length of the reference amino acid sequence and for failures of up to 5% in homology of the total number of amino acid residues in the reference sequence to be allowed. This aforesaid process for determining the percent identification between polypeptides may be applied to all proteins, polypeptides or a fragment thereof described in this invention.

In this application, antibodies are defined as being specifically bound if they bind to the antigen with a binding affinity (Ka) of 106 M-1 or higher, preferably 107 M-1 or higher, more preferably 108 M "1 or higher and even more preferably 109 M" 1 or higher. The affinity of an antibody can be readily determined by someone of ordinary skill in the art (e.g., by Scatchard analysis).

This invention provides compositions of the invention comprising: (a) a central particle with at least a first attachment site, wherein said central particle is a virus like particle (VLP) or a viral particle; and (b) at least one antigen with at least a second attachment site, wherein the at least single antigen is a Fel d1 protein, or fragment of Fel d1, and wherein (a) and (b) are attached. covalently by means of the at least one first attachment site and at least the second only attachment site. Preferably, a Fel d1 protein or a fragment of Fel d1 is attached to the central particle to allow an orderly and repeated array of VLP antigen to be formed. In preferably embodiments of the invention, at least 20, preferably at least 30, more preferably at least 60, and again, more preferably at least 120 and even more preferably at least 180 of Fel d1 protein or fragment thereof. Fel d1 are attached to the central particle.

Any virus known in the art which has an ordered and repeated structure can be selected as a VLP or a viral particle of the invention. Examples of DNA or RNA viruses, coat protein or capsid that can be used for the preparation of VLPs were set out at line 10-21 on page 25, line 11-28 on page 26 and line 4 on page 39 to line 4 of page 31 of WO 2004/009124. These exhibits are incorporated herein by reference.

Viruses or virus-like particles can be produced and purified from virus infected cell cultures. The resulting virus or virus-like particle for use in vaccines should preferably be non-replicating or non-infectious and more preferably non-replicating or infectious. UV radiation, chemical treatment, as with formaldehyde or chloroform, are the processes generally known to one of ordinary skill in the art to inactivate viruses. In one embodiment preferably, the central particle is a viral particle where preferably the viral particle is a viral particle of a bacteriophage and, even more preferably, said bacteriophage is a phage RNA and, even more preferably said phage RNA is an RNA selected from Q3, fr, GA or AP205.

In another preferred embodiment, the VLP is a recombining VLP. Virtually all commonly known viruses have been sequenced and are readily available to the public. The gene encoding the coat protein can be easily identified by a skilled artisan. The preparation of VLPs by recombinant expression of the coat protein in a host is common knowledge of a skilled artisan.

In a preferred embodiment, the virus-like particle comprises, or alternatively comprises recombinant proteins, mutants or fragments thereof, from a virus selected from the group consisting of: a) phage RNA; b) bacteriophages; c) hepatitis B virus, preferably its capsid protein (Ulrich et al., Virus Res 50: 141-182 (1998)) or its surface protein (WO 92/11291); d) measles virus (Warnes, et al., Gene 160: 173-178 (1995)); e) Sindbis virus; f) rotavirus (US 5,071,651 and US 5,374,426); g) foot-mouth disease virus (Twomey, et al., Vaccine 13: 1603 1610, (1995)); h) Norwalk virus (Jiang, X. et al., Science 250: 1580 1583 (1990), Matsui, S.M., et al., J. Clin). Invest. 87: 1456 1461 (1991); (i) alphavirus; j) retrovirus, preferably its GAG protein (WO 96/30523); k) retrotransposon Ty, preferably p1 protein; I) human papilloma virus (WO 98/15631); m) polyoma virus; n) tobacco mosaic virus and o) Flock House virus.

In one preferred embodiment, the VLP comprises or consists of more than one amino acid sequence, preferably two amino acid sequences of the recombinant proteins, mutants or fragments thereof. VLP comprising or consisting of more than one amino acid sequence is referred to in this application as mosaic VLP. The term "recombinant protein fragment" or the term "coat protein fragment" as used herein is defined as a polypeptide having at least 70%, preferably at least 80%, more preferably at least 90% and even more preferably at least 95% of the extent of the recombinant natural-type protein or coat protein, respectively, and which preferably retains the ability to form VLP. Preferably the fragment is obtained by at least one internal deletion, at least one truncation or at least a combination thereof. The term "recombinant protein fragment" or "coat protein fragment" will further encompass polypeptide having at least 80%, preferably 90% and even more preferably 95% of amino acid sequence identification with the "protein fragment". recombinant "or" fragment of a coat protein "as defined above and preferably capable of being assembled into a virus-like particle.

The term "mutant recombinant protein" or the term "recombinant protein mutant" as used interchangeably in this invention, or the term "mutant coat protein" or the term "mutant coat protein" as used interchangeably in this invention , refer to a polypeptide having an amino acid sequence derived from the recombinant natural-type protein or coat protein, respectively, wherein the amino acid sequence is at least 80%, preferably at least 85%, 90. %, 95%, 97% or 99% identical to the natural-type sequence and preferably have the ability to be mounted on a VLP.

In a preferred embodiment, the virus-like particle of the invention is hepatitis B virus. The preparation of hepatitis B virus-like particles has been disclosed, among others, in WO 00/32227, WO 01/8508 and WO 02/056905. All three documents are explicitly incorporated herein by reference. Other variants of HbcAg suitable for practical use of the present invention have been set forth on page 34-39 of WO 02/056905.

In still other preferred embodiments of the invention, a lysine residue is introduced into the HBcAg polypeptide to mediate the binding of Fel d1 of the invention to HBcAg VLP. In preferred embodiments, the VLPs and compositions of the invention are prepared using an HBcAg comprising, or alternatively consisting of amino acids 1-144 or 1-149, 1-185 of SEQ ID NO: 20 which is modified so that amino acids at positions 79 and 80 are replaced by a peptide having the amino acid sequence of Gli-Gli-üs-Gli-Gly. This change changes SEQ ID NO: 20 to SEQ ID NO: 21. In still other preferred embodiments, the cysteine residues at positions 48 and 110 of SEQ ID NO: 21, or their corresponding fragments, preferably 1-144 or 1-149, mutate to serine. The invention further includes compositions comprising Hepatitis B core mutant proteins which have the corresponding amino acid changes, noted above. The invention further includes compositions and vaccines, respectively, comprising HbcAg polypeptides, which comprise or alternatively consist of amino acid sequences that are at least 80%, 85%, 90%, 95%, 97% or 99%. identical to SEQ ID NO: 21.

In a preferred embodiment of the invention, the virus-like particle of the invention comprises or is essentially or alternatively comprised of recombinant coat proteins, mutants or fragments thereof, of a phage RNA. Preferably the phage RNA is selected from the group consisting of a) bacteriophage Q (3; b) bacteriophage R17; c) bacteriophage fr; d) bacteriophage GA; e) bacteriophage SP; f) bacteriophage MS2; g) bacteriophage M11; h) bacteriophage MX1; i) bacteriophage NL95; k) bacteriophage f2; I) bacteriophage PP7 and m) bacteriophage AP205.

In a preferred embodiment of the invention, the composition comprises a phage RNA coat protein, or mutants or fragments thereof, wherein the coat protein has an amino acid sequence selected from the group consisting of (a) SEQ IDNO: 1 ; referring to Qp CP; (b) a mixture of SEQ ID NO: 1 and SEQ ID NO: 2 (referring to protein Qp A1); (c) SEQ ID NO: 3; (d) SEQ ID NO: 4; (e) SEQ ID NO: 5; (f) SEQ ID NO: 6, (g) a mixture of SEQ ID NO: 6 and SEQ ID NO: 7; (h) SEQ ID NO: 8; (i) SEQ ID NO: 9; (j) SEQ ID NO: 10; (k) SEQ ID NO: 11; (I) SEQ: 12, (m) SEQ: 13, and (n) SEQ: 14.

In a preferred embodiment of the invention, the VLP is a mosaic VLP comprising or alternatively consisting of more than one amino acid sequence, preferably two protein coatings, or mutants or fragments thereof, of coatings. a phage RNA.

In a most preferred embodiment, the VLP comprises or alternatively consists of two different coat proteins from a phage RNA, said two coat proteins having an amino acid sequence of SEQ ID NO: 1 and SEQ ID NO: 2, or SEQ ID NO: 6 and SEQ ID NO: 7.

In a preferred embodiment of the invention, the virus-like particle of the invention comprises, is essentially constituted or, alternatively, consists of recombinant coat proteins, or mutants or fragments thereof, of bacteriophage RNA Qp, fr, AP205 or GA. In a preferred embodiment, the VLP is from a bacteriophage RNA, preferably from a Qp, fr, AP205 or GA bacteriophage RNA.

In a preferred embodiment, the VLP of the invention is a phage Qp RNA VLP. The Qp virus-like capsid or particle exhibited a phage capsid-like icosahedral structure with a diameter of 25 nm and T = 3 of near symmetry. The capsid contains 180 copies of the coat protein, which are linked in disulfide bridged pentamers and covalent hexamers (Golmohammadi, R. et al., Structure 4: 543-5554 (1996)), leading to remarkable stability of the capsid. Qp Capsids or VLPs constructed from recombinant Qp coat protein may, however, contain subunits that are not linked via disulfide to other subunits in the capsid, or are not fully bound. The Qp capsid or VLP exhibits unusual resistance to organic solvents and denaturing agents. Surprisingly, it was observed that concentrations of DMSO and acetonitrile at concentrations as high as 30% and guanidine concentrations as high as 1 M do not affect capsid stability. The high stability of the Q (3) capsid or VLP is an advantageous feature, particularly for its use in immunization and vaccination of mammals and humans according to the present invention.

Other virus-like particles of phage RNA, particularly Qp and fr, according to this invention are set forth in WO 02/056905, the disclosure of which is incorporated herein by reference in its entirety. Example 18 mainly of WO 02/056905 provides a detailed description of the Qp VLP particle preparation.

In a preferred embodiment, the VLP of the invention is an AP205 phage RNA VLP. Competently assembled mutant forms of AP205 VLPs, including amino acid 5 proline substituted AP205 coat protein for threonine, may also be used in the practice of the invention and lead to other preferred embodiments of the invention. WO 2004/007538 describes, particularly in Example 1 and Example 2, how to obtain VLP comprising AP205 coat proteins and, for the present, expression and purification thereof. WO 2004/007538 is incorporated herein by reference. AP205 VLPs are highly immunogenic and may be linked to Fel d1 of the invention to typically and preferably generate vaccine constructs displaying the repetitively oriented Fel d1 of the invention. Extract A high antibody titer is extracted with the thus exhibited Fel d1 of the inventions, demonstrating that the Fel d1 of the inventions provide access for interaction with antibody molecules and are immunogenic.

In a preferred embodiment, the VLP of the invention comprises or is comprised of a mutant virus coat protein, preferably a phage RNA, wherein the mutant coat protein has been modified by the removal of at least one lysine residue by replacement and / or by exclusion. In a preferred embodiment, the inventive VLP comprises, or is comprised of, a virus mutant coat protein, preferably a phage RNA, wherein the mutant coat protein has been modified by the addition of at least one lysine residue per replacement and / or by exclusion. The exclusion, substitution or addition of at least one lysine residue allows the level of coupling to be varied, that is, the amount of Fel d1 of the invention by VLP subunits of a virus, preferably RNA phages, particularly to combine and meet vaccine requirements.

In a preferred embodiment, the compositions and vaccines of the invention have an antigen density of 0.5 to 4.0. The term "antigen density" as used herein refers to the average number of Fel d1 of the invention, subunit linked, preferably by coat protein, of VLP, and hence preferably VLP of a phage RNA . Thus, this value is calculated as an average over all VLP subunits or monomers, preferably phage RNA VLP, in the composition or vaccines of the invention.

QP coat protein VLPs or capsids exhibit a defined number of lysine residues on their surface, with a defined topology of three lysine residues pointing towards the interior of the capsid and interacting with RNA, and four other exposed lysine residues to the outside of the capsid. Preferably, the at least first attachment site is a lysine residue, pointed at or on the outside of the VLP.

Qp mutants, of which lysine residues are replaced by arginines may be used for the present invention. Thus, in another preferred embodiment of the present invention, the virus-like particle comprises, is essentially constituted or, alternatively, is constituted by mutant Qp coat proteins. Preferably these mutant coat proteins comprise or alternatively comprise an amino acid sequence selected from the group of a) Qp-240 (SEQ ID NO: 15, Lis13-Arg of SEQ ID NO: 1) b) Qp-243 ( SEQ ID NO: 16, Asn10-Lys of SEQ ID NO: 1); c) Qp-250 (SEQ ID NO: 17, Lys2-Arg of SEQ ID NO: 1) d) Qp-251 (SEQ ID NO: 18, Lis16-Arg of SEQ ID NO: 1); ee) Qp-259 "(SEQ ID NO: 19, Lis2-Arg, Lis16-Arg of SEQ ID NO: 1. The construction, expression and purification of Qp coat protein VLPs and capsids, Qp mutant coat protein respectively given above are described in WO 02/056905. Particularly referred to herein is Example 18 of the above application.

In another preferred embodiment of the present invention, the virus-like particle comprises or, alternatively, is essentially constituted, or alternatively is constituted by mutant Qp coat protein, or mutants or fragments thereof, and the corresponding A1 protein. In yet another preferred embodiment, the virus-like particle comprises or alternatively is essentially constituted or alternatively is comprised of the amino acid sequence mutant coat protein of SEQ ID NO: 15, 16, 17, 18 or 19 and by the corresponding A1 protein.

Other phage RNA coat proteins have also been shown to self-assemble upon expression in a bacterial host (Kastelein, RA. Et al., Gene 23: 245-254 (1983), Kozlovskaya, TM. Et al., Dokl. Akad. Nauk SSSR 287: 452-455 (1986), Adhin, MR et al., Virology 170: 238-242 (1989), Priano, C. et al., J. Mol. Biol. 249: 283-297 (1995). )). In particular, the biological and biochemical properties of GA (Ni, CZ., Et al., Protein Sci. 5: 2485-2493 (1996), Tars, K et al., J. Mol.Biol. 271: 759- 773 (1997)) and de fr (Pushko P. et al., Prot. Eng. 6: 883-891 (1993), Liljas, L. et al. J Mol. Biol. 244: 279-290 (1994)) . The crystal structure of various bacteriophage RNAs was determined (Golmohammadi, R. et al., Structure 4: 543-554 (1996)). Using this information, exposed surface residues can be identified and thus phage RNA coat proteins can be modified such that one or more reactive amino acid residues can be inserted by insertion or substitution. Another advantage of phage RNA-derived VLPs is the high level of expression obtained in bacteria, enabling the production of large amounts of material at an affordable cost.

In one preferred embodiment, the composition of the invention comprises at least one antigen, wherein said at least one antigen is a Fel d1 protein.

In another preferred embodiment, Fed d1 protein comprises or alternatively comprises naturally occurring Fel d1. The primary structure of chain 1 is the sequence of SEQ ID NO 22.. Reported variants of chain 1 are Lis29-Arg or Asn, Va133-Ser, Val60-Leu. The primary structure of chain 2 is the sequence of SEQ ID NO 23, 25 or 26. Reported variants of chain 2 are Cis7-Fe, Fe15-Tr, Asn19-Ser, GH20-Leu, Ile55-Val, Arg57-Lis, Val58. -Fe of SEQ ID NO: 23, 25 or 26. Other variants of chain 2 are Glu69-Val, Tir70-Asp, Met72-Tr, Gln-77-Glu and Asn86-Lys of SEQ ID NO: 25; Met74-Tr, Gln79-Glu and Asn88-Lys of SEQ ID NO: 23. (Griffith IJ et al, Gene 113: 263-268 (1992); Morgenstern JP et al., Proc. Natl. Acad. Sci. USA 88: 9690-9694 (1991) Duffort OA, et al. Mol. Immunol. 28: 301 -309 (1991); Leitermann K., et al., J. Allergy Clin. Immunol., 74: 147-153 (1984); Kristensen, A.K., et al. (1997) Biol Chem 378, 899-908). Naturally occurring Fel d1 is obtained by purifying, for example, cat saliva, cat dandruff, household dust from a house where a cat lives, etc.

In a preferred embodiment, the Fel dt of the invention is a recombinant Fel d1 protein or a recombinant Fel d'1 fragment. Fel d1 recombinant protein or Fel d 1 recombinant fragment as used herein refers to a Fel d1 protein or a Fel d1 fragment that is obtained by a process comprising at least one step of the recombinant protein of DNA The terms "recombinant Fel d1 protein or recombinant Fel d1 fragment" and "recombinant produced Fel d1 protein or recombinant produced Fel d1 fragment" are used interchangeably herein and should have the same meaning. Recombinant protein Fel d1 or fragment thereof may be produced in prokaryotic expression systems such as E. coli (WO 2004/094639) or eukaryotic expression systems such as baculovirus (WO 00/20032) Seppálá et al simultaneously exposed expression of Fel 1 chain 1 and chain 2 by baculovirus bicistronic promoter (J.Biol. Chem. Nov, 2004). Fel d1 protein or fragment, produced by recombination, may be by glycosylation or not, depending on the host cell used to produce the recombinant protein.

In one embodiment, the recombinant Fel d1 protein comprises or alternatively comprises Fel d1 chain 1 and Fel d1 chain 2, wherein said Fel d1 chain 1 is linked to Fel d1 chain 2 exclusively by ligation. non-covalent, such as hydrophobic interactions.

In one embodiment, the recombinant Fel d1 protein comprises or, alternatively, comprises Fel d1 chain 1 and Fel d1 chain 2, wherein said Fel d1 chain 1 is linked to Fel d1 chain 2 by at least one. covalent bond. In one preferred embodiment, at least one covalent bond is a non-peptide bond, wherein said non-peptide bond is a disulfide bond or wherein said non-peptide bond is a disulfide bond.

For example, Fel d1 strand 1 and Fel d1 strand 2 may be separately expressed and then combined under conditions that allow the correct disulfide bond formation as a process of repeated mixing.

Alternatively, Fel d1 chain 1 and Fel d1 chain 2 can be simultaneously expressed in a host, for example, by cloning the genes encoding Fel d1 chain 1 and Fel d1 chain 2, respectively, according to with two promoters in one plasmid. In eukaryotic expression systems, Fel d1 chain 1 and Fel d1 chain 2 can be transcribed into one mRNA and translated separately by internal ribosome entry site (IRES).

In a preferred embodiment, the Fel d1 protein comprises or alternatively comprises a fusion protein, wherein said fusion protein comprises Fel d1 chain 1 and Fel d1 chain 2. In one preferred embodiment, said Fel d1 chain 1 and said Fel d1 chain 2 are directly fused by a peptide bond, linking the N-terminus of one chain to the C-terminus of the other chain. In one preferred embodiment, said Fel d1 chain 1 and said Fel d1 chain 2 are directly fused via a spacer, linking the N-terminus of one chain to the C-terminus of the other chain. Preferably said spacer is 1-30, preferably 1-25, preferably 1-20, preferably 1-15, preferably 1-9, preferably 1-5 and preferably 1-3 amino acids. Alternatively, said spacer is 10-30, preferably 10-25, more preferably 10-20, more preferably 13-20, more preferably 15-20, more preferably 13-17 and most preferably 15-25. 17 amino acids. Preferably said spacer is an amino acid sequence of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid residues. In one preferred embodiment, said spacer has 15 amino acids. In yet another preferred embodiment said spacer is (GGGGS).

In one preferred embodiment, the fusion protein comprises an amino acid sequence selected from the group consisting of: (a) SEQ ID NO: 24; (b) SEQ ID NO: 54; (c) SEQ ID NO: 55; (d) SEQ ID NO: 56; and (e) SEQ ID NO: 57.

In one preferred embodiment, said Fel d1 strand 2 is fused by its C-terminus to the Fel 'dl strand 1 N-terminus, either directly or by means of a spacer.

In yet another preferred embodiment said Fel d1 protein comprises or alternatively comprises an amino acid sequence of SEQ ID NO: 24.

WO 2004/094639 has exposed a folded recombinant Fel d1 with molecular and biological properties similar to its natural specimen and specifically a synthetic genetic code for direct fusion of the N-terminal from chain 2 to chain 1 of Fel d1. Expression in E. coli resulted in a non-covalently bound homodimer with an apparent molecular weight of 30 kDa, defined by size exclusion chromatography, each 19177 Da subunit exhibiting a disulfide pattern identical to that found in natural Fel d1 and Fel d1 is the recombinant having folds. Recombinant Fel d1 reacted with IgE from serum from cat allergic patients in the same way as natural Fel d1. Thus, this Fel d1 fusion protein mimics the antigenicity of natural Fel d1.

In one preferred embodiment, Fel d1 chain 1 is fused by its C-terminus to the Fel d1 chain 2 N-terminus, either directly or by means of a spacer.

In a preferred embodiment, Fel d 1 chain 1 comprises or alternatively comprises a sequence of SEQ ID NO: 22 or a homologous sequence thereof, wherein said homologous sequence has an identification relative to SEQ ID NO. : Greater than 80%, preferably greater than 90%, or even more preferably greater than 95%.

In one preferred embodiment, Fel d 1 chain 2 comprises or alternatively comprises a sequence of SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 26 or a homologous sequence thereof, wherein said homologous sequence has an identification with respect to SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 26 greater than 80%, preferably greater than 90%, or even more preferably greater than 95%.

In a preferred embodiment, the Fel d1 protein is a recombinant Fel d1 protein, wherein at least one disulfide bond is undone, preferably by mutation, more preferably by conservative substitution, such as from Cys to Ser. two native Fel d 1 peptides were identified, namely, Cis3 (1) -Cis73 (2), Cis44 (1) -48Cis (2) and Cis70 (1) -Cis7 (2), suggesting an antiparallel orientation of peptides from Fel d1. (Kristensen, A. K., et al. (1997) Biol Chem 378, 899-908). In another preferred embodiment, a disulphide bond of the Fel d1 protein is disrupted. In yet another preferred embodiment, two of these Fel d1 protein disulfide bonds are disrupted.

And in yet another preferred embodiment, all three of these Fel d1 protein disulfide bonds are disrupted. In a preferred embodiment, the Cys70 of chain 1 is deleted or mutated. In another preferred embodiment, the Cys73 of the chain is deleted or mutated. In a preferred embodiment, said Fel d1 protein is a fusion protein comprising Fel d1 chain 1 and Fel d1 chain 2, fused directly or by means of a spacer, wherein all three of these disulfide bonds are disrupted. In another preferred embodiment, said Fel d1 protein, comprising or alternatively comprising an amino acid sequence according to SEQ ID NO: 24, 55 or 57, wherein at least one, preferably at least three, and even more preferably at least five cysteines are removed by substitution or deletion.

In one preferred embodiment, the antigen of the invention comprises or alternatively comprises a fragment of Fel d1. It is a known fact that possession of immunogenicity does not generally require complete extension of a protein and that a protein generally contains more than one antigenic epitope, that is, antigenic site. A fragment or short peptide may be sufficient to contain at least one antigenic site that can immunospecifically bind to an antibody or a T cell receptor in the context of an MHC molecule. Antigenic site or sites may be determined by some techniques generally known to the skilled person. This determination can be made by sequence alignment and structure prediction. By way of example, it is possible to predict possible α-propellers, turns, intra- and inter-chain disulfide bonds, etc., using a program such as Rasmol. It is also possible to predict sequences that are inserted into the molecule or sequences that are exposed on the surface of the molecule. Sequences exposed on the surface of the molecule are more likely to understand natural antigenic site (s) and thus are useful in inducing therapeutic antibodies. After a surface peptide sequence has been determined, the antigenic site in this sequence can be further defined, for example, by intense mutagenesis processes (such as alanine selection mutagenesis, Cunnin-gham BC, Wells J. Science 1989 Jun 2; 244 (4908): 1081-5). Briefly, amino acids in this sequence mutate one by one to alanine and amino acids whose alanine mutations show, respectively, reduced antibody binding capacity (cultured against the natural-type sequence), or total loss of binding capacity. , are possibly components of the antigenic site. Another process for determining antigenic site (s) is to generate overlapping peptides that cover the entire length of the Fel d1 sequence (Geysen, PNAS Vol 81: 3998-4002, (1984) and Slootstra, JW et al., ( 1996) Mol. Divers 1, 87-96).

In one preferred embodiment, the antigen of the invention comprises or alternatively comprises at least one, preferably at least two Fel d1 epitopes, more preferably at least one Fel d1 chain 1 epitope, and at least one epitope. at least one epitope derived from Fel d1 chain 2.

Fel d1 T-cell reactive epitopes have been mapped throughout the Fel d1 protein and have been exposed in prior art as in the fourth paragraph of column 14 of US 6120769 and in column 130 and 131 of US6025162, these exposures being incorporated herein by reference. . In one preferred embodiment, the Fel d1 T-cell epitope is selected from a group consisting of: SEQ ID NO: 27-32.

The present invention provides a process for producing the composition of the invention comprising (a) providing a VLP with at least one attachment site; (b) providing at least one antigen, wherein said antigen is a Fel d1 protein or a Fel d1 fragment, with at least a second attachment site; and (c) combining said VLP and said at least one antigen for producing said composition, wherein said at least one antigen and said VLP are linked by the first and second attachment sites. In one preferred embodiment, delivery of at least one antigen, that is, a Fel d1 protein or a Fel d1 fragment with at least one second attachment site occurs by expression, preferably by expression in a bacterial system, preferably in E. coli. Usually a stop codon (tag), such as His tag, Mie tag is added to facilitate the purification process. In yet another specific approach, Fel d1 fragments of less than 50 amino acids can be chemically synthesized.

In a preferred embodiment of the invention, the VLP with at least one first attachment site is linked to the Fel d1 of the invention with at least one second attachment site by at least one peptide bond. The Fel d1 encoding gene of the invention, preferably the Fel d1 fragment, more preferably a fragment of up to 50 amino acids and even more preferably less than 30 amino acids, is ligated within the in-frame, either internally or preferably to the N or C-terminus of the gene encoding the VLP coat protein. Embodiments of the use of the antigen of the invention for coat protein, or mutants or fragments thereof, for a coat protein of a virus have been described in line 20 of page 62 to line 17 of page 68 of WO 2004/009124, these being: incorporated herein by reference.

In one preferred embodiment, a Fel d fragment | is fused to the N or C-terminus of a coat protein, or mutants or fragments thereof, of AP205 phage RNA. In yet another preferred embodiment, the fusion protein further comprises a spacer, wherein said spacer is fused to the AP205 coat protein, or fragments or mutants thereof, and a Fel d1 fragment.

In a preferred embodiment of the present invention, the composition comprises or alternatively consists essentially of a virus-like particle with at least one first attachment site attached to at least one Fel d1 of the invention with at least a second site. fixing by means of at least one covalent bond, and preferably this covalent bond is a non-peptide bond. In a preferred embodiment of the present invention, the first attachment site comprises or preferably is an amino group and preferably the amino group is a lysine residue. In another preferred embodiment of the present invention, the second attachment site comprises or preferably is a sulfhydryl group and preferably a sulfhydryl group and a cysteine.

In a most preferred embodiment of the invention, the at least one first attachment site is an amino group, preferably an amino group of a lysine residue and the at least one second attachment site is a sulfhydryl group, preferably a sulfhydryl group of a cysteine.

In a preferred embodiment of the invention, the Fel d1 of the invention is bound to the VLP by means of a chemical crosslink, typically and preferably using a heterobifunctional crosslinker. In preferred embodiments, the heterobifunctional crosslinker contains a functional group which may react with the first preferred attachment sites, preferably with the amino group, more preferably with the VLP lysine residue (s) amino groups and yet another functional group. which may react with the second preferred attachment site, i.e. a sulfhydryl group, preferably cysteine residue (s), inherent or artificially added to the Fel d1 of the invention, and optionally also available for reduction reaction. Several heterobifunctional crosslinking are known in the art. They include the preferred crosslinkers SMPH (Pierce), Sulfo-MBS, Sulfo-EMCS, Sulfo-GMBS, Sulfo-SIAB, Sulfo-SMPB, Sulfo-SMCC, SVSB, SIA and other available crosslinkers, for example from Pierce Chemical. Company, and which have a reactive functional group for amino groups and a reactive functional group for sulfhydryl groups. All the above mentioned crosslinking leads to the formation of an amide bond upon reaction with the amino group and a thioether bond with the sulfhydryl groups. Another class of crosslinking suitable for the practice of the invention is characterized by the introduction of a disjoint bond between the Fel d1 of the invention and the VLP in the coupling. Preferably crosslinkers belong to this class and include, for example, SPDP and Sulfo-LC-SPDP (Pierce).

In a preferred embodiment, the composition of the invention further comprises a linker. The engineering of a second fixation site in the Fel d1 of the invention is achieved by combining a linker which preferably contains at least one suitable amino acid as a second fixation site according to the exposures of this invention. Accordingly, in a preferred embodiment of the present invention, a linker is associated with the Fel d1 of the invention by at least one covalent bond, preferably by at least typically one peptide bond. Preferably, the connector comprises or alternatively comprises the second attachment site. In yet another preferred embodiment, the linker comprises a sulfhydryl group, preferably from a cysteine residue. In another preferred embodiment, the amino acid linker is a cysteine residue.

Selection of a linker will depend on the nature of the Fel d1 of the invention, its biochemical properties such as p1, charge distribution and glycosylation. In general, flexible amino acid linkers are preferred. In yet another preferred embodiment of the present invention, the linker consists of amino acids, wherein most preferably the linker consists of a maximum of 25, preferably a maximum of 20 and even more preferably a maximum of 15 amino acids. In a once again preferred embodiment of the invention, the amino acid linker does not contain more than 10 amino acids. Preferred embodiments of the linker are selected from the group consisting of: (a) CGG; (b) N-terminal gamma 1 linker (e.g., CGDKTHTSPP, SEQ ID NO: 58); (c) N-terminal gamma 3 linker (e.g., CGGPKPSTPPGSSGGAP, SEQ ID NO: 69); (d) articulated regions of Ig; (e) N-terminal glycine linkers (e.g., GCGGGG, SEQ ID NO: 59); (f) (G) kC (G) n with n = 0-12 and k = 0-5; (g) N-terminal glycineserine linkers ((GGGGS) n, n = 1-3, with yet another cysteine, (e.g. SEQ ID NO: 60, which corresponds to one embodiment where n = 1); (h) (G) kC (G) m (S) 1 (GGGGS) n, with n = 0-3, k = 0-5, m = 0-10, 1 = 0-2 (e.g. SEQ ID NO: 61 which corresponds to an embodiment wherein n = 1, k = 1.1 = 1 and em = 1) (i) GGC (k) GGC-NH2 (I) C-terminal gamma 1 linker (e.g. DKTHTSPPCG (M) gamma C-terminal linker (e.g., PKPSTPPGSSGGAPGGCG, SEQ ID NO: 63); (n) glycine C-terminal linkers (GGGGCG, SEQ ID NO: 64); ) (G) nC (G) k, with n = 0-12 and k = 0-5, (p) C-terminal glycine-serine linkers ((SGGGG) n, n = 1-3 with an additional cysteine (e.g. SEQ ID NO: 65, which corresponds to one embodiment, where n = 1)); (q) (G) m (S) 1 (GGGGS) n (G) oC (G) k, with n = 0- 3, k = 0-5, m = 0-10, 1 = 0-2, and o = 0-8 (e.g. SEQ ID NO: 66, which corresponds to one embodiment, where n = 1, k = 1, 1 = 1, o = 1 and m = 1) In yet another preferred embodiment, the linker is added to the N-term. Fel d1 of the invention. In yet another preferred embodiment of the invention, the connector is added to the Fel d1 C-terminal of the invention.

Preferred linkers according to this invention are glycine (G) n linkers, further containing a cysteine residue as a second attachment site, such as the N-terminal glycine linker (GCGGGG) and C-terminal glycine linker (GGGGCG). Other preferred embodiments are the C-terminal glycine-lysine linker (GGKKGC, SEQ ID NO: 67) and N-terminal glycine-lysine linker (CGKKGG, SEQ ID NO: 68), GGCG to GGC or GGC-NH2 linkers ( "NH2" designating amidation) at the C-terminus of the peptide or CGG at its N-terminus. Generally, glycine residues will be inserted between bulk amino acids and cysteine to be used as the second fixation site to avoid steric hindrance of the bulk amino acid in the coupling reaction.

In one preferred embodiment, the linker is fused to the N-terminus of the Fel d1 protein or Fel d1 fragment. In yet another preferred embodiment, the linker is GCGG. In another preferred embodiment, the linker is fused to the C-terminus of the Fel d1 protein or Fel d1 fragment. In yet another preferred embodiment, the linker is GGC. In case the Fel d1 protein or Fel dl fragment consists of two polypeptide chains, this Fel d1 protein or Fel d1 fragment has two N-terminal moieties and two C-terminal moieties. The connector may be fused to the two N-terminal portions or the two C-terminal portions. Preferably, the linker is fused to only one of the two N-terminal portions or to only one of the two C-terminal portions.

Attachment of the Fel d1 of the invention to the VLP using a heterobifunctional crosslinker according to the preferred methods described above allows the coupling of the Fel d1 of the invention to the VLP in an oriented manner. Other methods of binding the Fel d1 of the invention to the VLP include processes wherein the Fel d1 of the invention is crosslinked to the VLP using carbodiimide EDC and NHS. The Fel d1 of the invention may also be initially thiolated by reaction, for example, with SATA, SATP or imino-thiolane. The Fel d1 of the invention, after the shield has been removed, can then be coupled to the VLP as follows. After separation of excess thiolation reagent, Fel d1 of the invention reacts with VLP, previously activated with a heterobifunctional crosslinker, comprising a reactive cysteine moiety and thus exhibiting at least one or more residue reactive functional groups. of cysteine, with which the thiolated Fel of the invention may react as described above. Optionally, low amounts of a reducing agent are included in the reaction mixture. In still other processes, the Fel d1 of the invention is attached to the VLP using a homobifunctional crosslinker such as glutaraldehyde, DSG, BM [PEO] 4, BS3, (Pierce) or other known homobifunctional crosslinking linkers with functional groups. reactive for amino groups or carboxyl groups of the VLP.

In still other embodiments of the present invention, the composition comprises or, alternatively, consists essentially of a Fel d1-linked virus-like particle of the invention by chemical interactions, wherein at least one of these interactions is not a covalent bond. . For example, the binding of the VLP to the Fel d1 of the invention may be effected by biotinylation of the VLP and expression of the Fel d1 of the invention as a streptavidin fusion protein.

One or more antigen molecules, that is, Fel d1 of the invention, may be attached to a VLP subunit, preferably phage RNA coat proteins, preferably by exposed lysine residues of phage RNA VLP coat proteins, if sterically possible. A specific feature of phage RNA VLPs, and particularly of Qp coat protein VLPs, is thus to enable the coupling of various antigens by subunit. This allows the generation of an antigen-filled arrangement.

In very preferred embodiments of the invention, the Fel d1 of the invention is bound via a cysteine residue which has been added to the N-terminal or the C-terminal of the Fel d1 of the invention, or natural cysteine residue contained in the Fel d1. of the invention to lysine residues of phage RNA VLP coat proteins and particularly to Qp coat protein.

As described above, there are four lysine residues exposed on the surface of the Qp coat protein VLP. Typically and preferably, these residues are derivatized by reacting with a crosslinker molecule. If not all exposed lysine residues can be coupled to an antigen, the lysine residues that reacted with the crosslinker are left with a crosslinker molecule attached to the e-amino group after the derivatization step. This leads to the disappearance of one or several positive charges and may impair the solubility and stability of the VLP. By replacing some of the lysine residues with arginines, as in the Qp coat protein mutants, we prevent excessive disappearance of positive charges since the arginine residues do not react with the crosslinkers preferably.

In addition, replacing lysine residues with arginine residues may lead to more defined antigenic arrangements, as fewer sites are available for reaction with the antigen.

In accordance with the foregoing, exposed lysine residues were replaced by arginines in the following Qp coat protein mutants: Qp-240 (Lis13-Arg; SEQ ID NO: 15), Qp-250 (Lis 2-Arg, Lis13-Arg SEQ ID NO: 17), Qp-259 (Lys 2-Arg, Lys16-Arg; SEQ ID NO: 19) and Qp-251; (Lis16-Arg, SEQ ID NO: 18). In yet another embodiment, we expose a mutant Qp coat protein with an additional Qp-243 lysine residue (Asn 10-Lys; SEQ ID NO: 16) suitable for obtaining higher density antigenic arrays.

In a preferred embodiment of the invention, the VLP of the invention is produced by recombination by a host wherein said VLP is essentially free of host RNA, preferably from host nucleic acids. In yet another preferred embodiment, the composition further comprises at least one linked polyanionic macromolecule, preferably packaged or inserted into the VLP. In yet another preferred embodiment, the polyanionic macromolecule is polygutamic acid and / or polyaspartic acid.

Essentially free from host RNA, preferably from host nucleic acids. The term "essentially free of host RNA, preferably from host nucleic acids" as used herein, refers to the amount of host RNA, preferably from host nucleic acids, comprised by the VLP, this amount being typically and preferably less than 30 æg, preferably less than 20 æg, more preferably less than 10 æg, even more preferably less than 8 æg, even more preferably less than 6 æg, even more preferably less than 4 æg and most preferably less than 2 æg per mg of VLP. Host, as used in the preceding context, refers to the host in which NPV is produced by recombination. Conventional methods for determining the amount of RNA, preferably nucleic acids, are known to the person skilled in the art. The typical and preferred method for determining the amount of RNA, preferably nucleic acids, according to the present invention is described in Example 17 of PCT / EP2005 / 055009, filed October 5, 2005, by the same designator. Similar, similar or analogous conditions are typically and preferably used for determining the amount of RNA, preferably nucleic acids, for compositions of the invention, comprising VLPs other than Qp. Modifications of the eventually necessary conditions are part of the knowledge of the skilled person. The numerical value of the amounts determined should typically and preferably be understood to comprise values that are within ± 10%, preferably within ± 5% of the indicated numerical value.

Polyanionic Molecule: The term "polyanionic macromolecule" as used herein refers to a relative high molecular weight molecule comprising negatively charged repeating groups, the structure of which essentially comprising the multiple repeating units that are actually derived. effectively or conceptually of molecules of low relative molecular weight. A polyanionic macromolecule should have a molecular weight of at least 2000 Dalton, more preferably at least 3000 Dalton and even more preferably at least 5000 Dalton. The term "polyanionic macromolecule" as used herein typically and preferably refers to a molecule that is not capable of activating Toll-like receptors. Thus, the term "polyanionic macromolecule" typically and preferably excludes Toll-like receptor ligands and even more preferably excludes immunostimulant substances such as Toll-like receptor ligands, immunostimulant nucleic acids and lipopolysaccharides (LPS). More preferably, the term "polyanionic macromolecule" as used herein refers to a molecule that is not capable of inducing cytokine production. Even more preferably, the term "polyanionic macromolecule" excludes immunostimulatory substances. The term "immunostimulant substance" as used herein refers to a molecule that is capable of inducing and / or enhancing immune response specifically against the antigen comprised in the present invention.

Host RNA, preferably host nucleic acids: The term "Host RNA, preferably host nucleic acids" or the term "Host RNA, preferably secondary structure host nucleic acids", as used herein, refers to RNA, preferably nucleic acids that are originally synthesized by the host. RNA, preferably nucleic acids, may, however, undergo chemical and / or physical changes during the process to reduce or suppress the amount of RNA, preferably nucleic acids, typically and preferably by the processes of the invention, for example. For example, the size of RNA, preferably nucleic acids, may be shortened or the secondary structure may be altered. However, even this RNA or resulting nucleic acids are still considered host RNA or host nucleic acids.

Methods for determining the amount of RNA and reducing the amount of RNA comprised by NPV were described in PCT / EP2005 / 055009, filed by the same designator on October 5, 2005, and thus the entire order is here. incorporated by reference. Reducing or eliminating the amount of host RNA, preferably host nucleic acid, minimizes or reduces unwanted T cell responses, such as R cell inflammatory response and T cell cytotoxic response, and other unwanted side effects, such as fever, while maintaining strong antibody response specifically against Fel d1.

In a preferred embodiment, this invention provides a process for preparing the inventive and VLP compositions of a bacteriophage RNA - Fel d1 of the invention, wherein said VLP is produced by recombination by a host and wherein said VLP is essentially free of host RNA, preferably from host nucleic acids, comprising the steps of: a) recombinant production of a virus-like particle (VLP) with at least a first attachment site by a host, wherein the said VLP comprises coat proteins, variants or fragments thereof of a bacteriophage RNA; b) disassembling said virus-like particle into said coat proteins, variants or fragments thereof, from said bacteriophage RNA; c) purifying said coat proteins, variants or fragments thereof from said bacteriophage RNA to a virus-like particle, wherein said virus-like particle is essentially free of host RNA, preferably host nucleic acids; and e) ligating at least one antigen of the invention with at least one second attachment site with said VLP obtained from step (d). In yet another preferred embodiment, reassembly of said purified coat proteins, variants or fragments thereof is effected in the presence of at least one polyanionic macromolecule.

In a preferred embodiment, the composition of the invention further comprises at least one immunostimulating substance capable of inducing and / or enhancing an immune response. Preferably the immunostimulant substance is a Toll-like receptor binder, preferably selected from the group consisting of: (a) immunostimulatory nucleic acids; (b) peptide glycans; (c) lipopolysaccharides; (d) lipoteonic acids; (e) imidazoquinolines compounds; (f) flagellins; (g) lipoproteins; (h) immunostimulating organic molecules; (i) oligonucleotides containing unmethylated CpG; and (j) any mixtures of (a), (b), (c), (d), (e), (f), (g), (h) and (i).

In yet another preferred embodiment, the immunostimulatory nucleic acid is preferably selected from the group consisting of: (a) a nucleic acid of bacterial origin; (b) a nucleic acid of viral origin;

(c) a nucleic acid comprising an unmethylated CpG motif; (d) a double stranded RNA; (e) a single stranded RNA; and (g) an unmethylated CpG motif-free nucleic acid. Immunostimulatory nucleic acids that do not contain unmethylated CpG motif have been described in the prior art, for example in WO01 / 22972. The term "nucleic acid" as used herein refers to a molecule composed of covalently linked linear monomers (nucleotides). It indicates a molecular chain of nucleotides and does not refer to a specific product extension. Thus, oligonucleotides are included in the definition of nucleic acid. The nucleotide bond is typically and preferably a phosphodiester bond. Also encompassed by this invention are nucleic acids comprising binding modifications such as, for example, a phosphorothioate bond.

In a preferred embodiment, the immunostimulant substance is mixed with the recombinant VLP. In yet another preferred embodiment, the immunostimulant substance is preferably attached to the VLP of the invention.

Detailed descriptions of immunostimulant substance, particularly immunostimulatory nucleic acid, more particularly oligonucleotides comprising unmethylated CpG have been set forth in WO 03/024480, 03/024481 and PCT / EP / 04/003165. Methods for mixing immunostimulating substances with NPV-antigen have been disclosed in WO 03/024480. Methods of packaging immunostimulant substances within the VLP have been disclosed in WO 03/024481. WO 03/024480, 03/024481 and PCT / EP / 04/003165 in their entirety are hereby incorporated by reference. VLP can usually induce and / or enhance the immune system. However, the term "immunostimulant substance" as used in the context of this application refers to an immunostimulant substance other than the VLP used for the compositions of the invention rather than being added to said VLP.

The inclusion of immunostimulatory substances, preferably immunostimulatory nucleic acids, in the composition of the invention may direct immune responses to Th1 responses and thereby suppress Th2 responses.

In one feature, the invention provides a vaccine comprising the composition of the invention. In one feature, the invention provides a vaccine comprising the composition of the invention and a suitable buffer. In a preferred embodiment, the vaccine composition further comprises an adjuvant. Administration of at least the only adjuvant may therefore be present before, simultaneously or after administration of the composition of the invention. The term "adjuvant" as used herein refers to non-specific immune response stimulants that allow the generation of a host accumulation which, when combined with the vaccine and a pharmaceutical composition, respectively, of the present invention can provide an even more enhanced immune response.

In another preferred embodiment, the vaccine composition is devoid of adjuvant. An advantageous feature of the present invention is the high immunogenicity of the composition even in the absence of adjuvants. Avoided use of adjuvants may reduce the possible occurrence of side effects related to the use of adjuvants. Accordingly, administration of the vaccine of the invention to a patient will preferably occur without the administration of at least one adjuvant to the same patient before, simultaneously or after administration of the vaccine.

The invention further describes a method for immunization comprising administering the vaccine of the present invention to a human or non-human mammal, such as a dog or cat. Without wishing to limit the present invention by theory, injecting the vaccine composition into a cat may neutralize Fel d1 and thus reduce the amount of Fel d1 in the cat's saliva.

The vaccine may be administered by a variety of methods known in the art, but will normally be administered by injection, infusion, inhalation, oral administration or other suitable physical modes. The conjugates may alternatively be administered intramuscularly, intravenously, transmucosally, transcutaneously, intranasally, intraperitoneally or subcutaneously. Conjugate components for administration include aqueous (e.g., physiological saline) and non-aqueous sterile solutions and suspensions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Occlusive vehicles or dressings may be used to increase skin permeability and improve antigen-absorption.

Vaccines of the invention are considered to be "pharmacologically acceptable" if their administration can be tolerated by an individual container. In addition, the vaccines of the invention will be administered in a "therapeutically effective amount" (that is, an amount that produces a desired physiological effect). The nature or type of immunological response is not a limiting factor of this exposure. Without wishing to limit the present invention by explaining the following mechanism, the vaccine of the invention may induce antibodies, presumably IgG subtypes, which bind to Fel d1, thereby preventing Fel d1 from being seen by mast cell and basophil-bound IgE. Alternatively or simultaneously, the composition of the present invention directs immune responses to Th1 responses, suppressing the development of Th2 responses and therefore the production of IgE antibodies, a major component in allergic reactions.

In one aspect, the invention provides a pharmaceutical composition comprising the composition as described in the present invention and an acceptable pharmaceutical carrier. When the vaccine of the invention is administered to an individual, it may be in a form containing salts, buffers, adjuvants or other substances that are suitable for enhancing the effectiveness of the conjugate. Examples of materials suitable for use in the preparation of pharmaceutical compositions are provided from a variety of sources, including Remington's Pharmaceutical Sciences (Osol, A, ed., Mack Publishing Co., (1990)).

The invention teaches a process for producing the composition of the invention comprising the steps of: a) providing a central particle with at least one attachment site, wherein said central particle is a virus-like particle or a viral particle; (b) providing at least one antigen, wherein said antigen is a Fel d1 protein or a Fel d1 fragment, with at least a second attachment site; and (c) combining said central particle and said at least one antigen for producing said composition wherein said at least one antigen and said VLP are linked by the first and second attachment sites. .

In another preferred embodiment, the core particle delivery step, with at least a single first attachment site, further comprises the steps: (a) disassembling said core particle for coat proteins, mutants or fragments thereof, of said central particle; (b) purifying said coat proteins, mutants or fragments thereof; (c) reassembling said purified coat proteins from said central particle, wherein said central particle is essentially free of host RNA, preferably host nucleic acids. In yet another preferred embodiment, reassembly of said purified coat proteins is effected in the presence of at least one polyanionic macromolecule or at least one immunostimulatory nucleic acid.

In one aspect, the invention provides a method of using the compositions of the invention for preventing and / or treating cat allergy in a mammal, wherein said mammal is preferably a human or a dog.

In one aspect, the invention discloses use of the composition of the invention as a medicament. In another aspect, the invention provides the use of the composition of the invention for the manufacture of a medicament for treating cat allergy in a mammal, wherein said mammal is preferably a human or dog.

In one aspect, this invention provides a Fel d1 fusion protein comprising Fel d1 chain 1 and Fel d1 chain 2 fused by an amino acid spacer that connects the N-terminus of a chain to the C-terminus. of the other chain, wherein said spacer comprises an amino acid sequence of 10-30, preferably 10-25, preferably 10-20, preferably 13-20, preferably 15-20, preferably 13-17 and preferably 15-17 wherein said fusion protein is not a fusion protein comprising strand 1 of SEQ ID NO: 22 fused through (GGGGS) 3 to the N-terminus of strand 2 of SEQ ID NO: 23, 25 or 26, and wherein said declined fusion protein is expressed in baculovirus expression system.

WO2004094639 describes a Fel d1 fusion protein by linking chain 1 and chain 2 with a linker selected from a carbon-nitrogen bond and a short peptide, i.e. from 1 to 9, preferably from 1 to 5, and particularly preferable. from 1 to 3 amino acids. He further argues that surprisingly a short peptide or binding does not induce relevant restrictions or offsets. However, WO2004094639 fails to mention a linker longer than 9 amino acids.

WO 00/20032 describes recombinant baculovirus-expressed Fel D1, comprising chain 1 and chain 2, expressed in series and joined by a glycine / serine linker (GGGGS) 3. WO 00/20032 also reported that rFel d1 immunoreactivity to IgG and IgE antibody is significantly enhanced by the expression of the allergen in baculovirus compared to the allergen expressed in E. coli. The Fel d1 fusion protein of the invention may be produced in

a prokaryotic expression system or a eukaryotic expression system such as a baculovirus system. In one preferred embodiment, said Fel d1 fusion protein of the invention is produced from E. coli.

In a preferred embodiment, the spacer, comprised of the Fel d1 fusion protein, is comprised of an amino acid sequence of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 residues. amino acids.

In a preferred embodiment, the spacer comprised of the Fel d1 fusion protein consists of an amino acid sequence with 2, 3 or 4 four GGGGS repeats.

In a preferred embodiment, Fel d1 chain 2 is at the N-terminus of the fusion protein of the invention.

In another preferred embodiment, Fel d1 strand 1 is at the N-terminus of the fusion protein of the invention. In another preferred embodiment, the fusion protein is produced from E. coli.

In a most preferred embodiment, the Fel d1 fusion protein of the invention comprises an amino acid sequence selected from the group consisting of: (a) SEQ ID NO: 54; (b) SEQ ID NO: 55; and (c) SEQ ID NO: 57. The invention further provides the nucleotide sequence encoding the Fel d1 fusion protein of the invention. In one preferred embodiment, the invention provides a nucleotide sequence encoding a fusion protein of the invention selected from the group consisting of: (a) SEQ ID NO: 54; (b) SEQ ID NO: 55; and (c) SEQ ID NO: 57. In yet another preferred embodiment, the Fel d1 fusion protein of the invention is produced in E. coli. In another preferred embodiment, the Fel d1 fusion protein of the invention comprises or alternatively comprises an amino acid sequence of SEQ ID NO: 57, wherein said fusion protein of the invention is produced in E. coli. .

In one aspect, the invention provides a use of the Fel d1 fusion protein of the invention for diagnosis and treatment of cat allergy.

EXAMPLES EXAMPLE 1

Preparation of Qp VLPs of the invention by disassembly / reassembly in the presence of different polyanionic macromolecules, resulting in reassembled Qp VLPS.

(A) Disassembly of Prior Art Qp VLP 45 mg Prior Art Qp VLP (2.5 mg / ml as determined by Bradford analysis) in PBS (20 mM Phosphate, 150 mM NaCl NaCl, pH 7 , 5), purified from E. coli lysate, was reduced with 10 mM DTT for 15 min at room temperature with stirring. Magnesium chloride was then added to a final concentration of 0.7 M and incubation continued for 15 min at room temperature, with shaking, leading to precipitation of the encapsulated host cell RNA. The solution was centrifuged for 10 min at 4000 rpm at 4 ° C (Eppendorf 5810 R in A-4-62 fixed angle rotor used in all subsequent steps) to remove precipitated RNA from the solution. The supernatant containing the released dimeric Qp coat protein was used for chromatographic purification steps.

(B) Purification of Qp coat protein by cation exchange chromatography and size exclusion chromatography. The disassembly reaction supernatant containing the dimeric coat protein, host cell proteins, and host cellular RNA residues was diluted 1:15 in water to adjust conductivity below 10 ms / cm 2 and injected into an SP-Sepharose FF column (xk16 / 20.6 ml, Amersham Bioscience). The column was equilibrated beforehand with 20 mM sodium phosphate buffer, pH7. Elution of the bound coat protein was achieved by step gradient 20 mM sodium phosphate / 500 mM sodium chloride, and the protein was collected on a volumede. fraction of approximately. Chromatography was conducted at room temperature with a flow rate of 5 ml / min, and absorbance was monitored at 260 nm to 280 nm.

In the second step, the isolated Qp coat protein (the eluted fraction of the cation exchange column) was loaded (in two steps) on a Sefacril S-100 HR column (xk26 / 60, 320ml, Amersham Bioscience), e-calibrated with 20 mM sodium phosphate / 250 mM sodium chloride, pH 6.5. Chromatography was conducted at room temperature with a flow rate of 2.5 ml / min, and absorbance was monitored at 260 nm to 280 nm. Five ml fractions were collected.

(C1) Reassembly of QLP VLP by dialysis Purified Qp coating protein (2.2 mg / ml in 20 mM sodium phosphate, pH 6.5), a polyanionic macromolecule (2 mg / ml in water), urea (7.2 M in water) and DTT (0.5 M in water) were mixed to final concentrations of 1.4 mg / ml coat protein, 0.14 mg / ml of corresponding macromolecule, 1 M urea and 2.5 mM DTT. The mixtures (1 ml each) were dialyzed for two days at 59 ° C in 20 mM TrisHCL, 150 mM Na-Cl, pH 8 using 3.5 kDa bound membranes. The polyanionic molecules were: polygalacturonic acid (25000-50000, Fluka), dextran sulfate (PM 5000 and 10000, Sigma), poly-L-aspartic acid (PM 11000 and 33400, Sigma), poly-L-glutamic acid (PM 3000 , 13600 and 84600, Sigma) and yeast and wheat germ tRNAs.

(C2) Reassembly of Qp VLP by diafiltration 33 ml of purified Qp coat protein (1.5 mg / ml in 20 mM sodium phosphate, pH 6.5, 250 mM NaCl) were mixed with water and urea (7 2 mM in water), NaCl (5 M in water) and poly-L-glutamic acid (2 mg / ml in water, MW: 84600). The volume of the mixture was 50 ml and the final component concentrations were 1 mg / ml coat protein, 300 mM NaCl, 1.0 mM urea and 0.2 mg / ml poly-L-glutamic acid. The mixture was then diafiltered at room temperature against 500 ml of 20 mM TrisHCl, pH8, 50 mM NaCl, a cross flow rate of 10 ml / min and a flow permeability rate of 2.5 ml / min. in a tangential flow filtration apparatus using a Pellicon XL membrane housing (Biomax 5K, Millipore).

EXAMPLE 2

In Vitro Assembly of AP205 NPVs 5 (A) AP205 Coating Protein Purification Disassembly: 20 ml solution containing AP205 VLP (1.6 mg / ml in PBS, purified E.coli extract) was mixed with 0.2 ml 0.5 M DTT and incubated for 30 min at room temperature. 5 ml of 5 M NaCl was added and the mixture was then incubated for 15 min at 60 ° C causing DTT-reduced coating proteins to precipitate. The cloudy mixture was centrifuged (Sorvall SS34 rotor, 10,000 g, 10 min, 20 ° C) and the supernatant was discarded and the pellet dispersed in 20 ml 1 M urea / 20 mM NA Citrate, pH 3.2. . After stirring for 30 min at room temperature, the dispersion was adjusted to pH 6.5 by the addition of 1.5 M Na 2 HPO 4 and then centrifuged (Sorvall SS34 rotor, 10,000 g, 10 min, 20 ° C) to obtain a supernatant containing dimeric coating protein.

Cation Exchange Chromatography: The supernatant (see above) was diluted with 20 ml of water to adjust conductivity to approximately 5 ms / cm. The resulting solution was injected into a 6 ml SP Sepharose FF (Amersham Bioscience) column, previously equilibrated with 20 mM sodium phosphate buffer, pH 6.5. After loading, the column was washed with 48 ml of 20 mM sodium phosphate buffer, pH 6.5, followed by elution of the linear gradient bound coating protein to 1 M NaCl over 20 column volumes. Main peak fractions were pooled and analyzed by SDS-PAGE and UV spectroscopy. According to SDS-PAGE analysis, the isolated coat protein was essentially pure from other protein contamination. According to UV spectroscopy, the protein concentration was 0.6 mg / ml (total amount: 12 mg), whereas 1 unit of A280 reflects 1.01 mg / ml of AP205 coat protein. In addition, the value of A280 (0.5999) over the value of A260 (0.291) is 2, indicating that the preparation is essentially free of nucleic acids. (B) AP205 VLP Assembly

Mounting in the absence of any polyanionic macromolecule: The protein fraction eluted from the previous step was diafiltered and concentrated by TFF to a protein concentration of 1 mg / ml in 5 20 mM sodium phosphate, pH 6.5. 500 µl of this solution was mixed to 50 µl of a 5 M NaCl solution and incubated for 48 h at room temperature. Formation of reassembled VLPs in the mixture was shown by non-reducing SDS-Page and size exclusion HPLC. A TSKgel G5000 PWXL (Tosoh Bioscience) column, equilibrated with 20 mM sodium phosphate, 150 mM NaCl, pH 7.2, was used for HPLC analysis.

Mounting in the presence of polyglutamic acid: 375 æl purified AP205 coat protein (1 mg / ml in 20 mM sodium phosphate, pH 6.5) were mixed with 50 æl stock NaCl solution (5 M in water), 50 µl of polyglutamic acid stock solution (2 mg / ml in water, MW: 86400, Sigma) and 25 µl of water. The mixture was incubated for 48 h at room temperature. Formation of reassembled VLPs in the mixture was shown by non-reducing SDS-Page and size exclusion HPLC. The coating protein in the mixture was almost completely incorporated into the VLPs, showing a higher assembly efficiency than that for the AP205 coating protein assembled in the absence of any polyanionic macromolecule.

EXAMPLE 3

Cloning of Fel d1 fusion proteins Genes encoding Fel d1 strand 1 and strand 2, respectively, were produced by PCR amplification using overlapping DNA primers as shown below. Forward (F) and reverse (R) initiators are indicated. The fragments were ligated into a pCRI1-TOPO vector (Invitrogen) and transformed into XL1-Blue. The sequence of insertions of chain 1 and chain 2 of Fel d1 was verified. Initiate SequenceMF: SEQ ID NO: 34;

Primer String 2R: SEQ ID NO: 35

3F Primer Sequence: SEQ ID NO: 36 4R Primer Sequence: SEQ ID NO: 37 5F Primer Sequence: SEQ ID NO: 38 6R Primer Sequence: SEQ ID NO: 39 7F Primer Sequence: SEQ ID NO: 40 ID 8R: SEQ ID NO: 41

9F Primer Sequence: SEQ ID NO: 42 10R Primer Sequence: SEQ ID NO: 43 Fel Fusion Constructs d1:

FELD1 refers to the N-terminal strand 2 protein fused directly to strand 1. The nucleotide sequences encoding FELD 1 were created by the PCR-splicing overlap extension (SOE) technique using primer linker 11 (SEQ ID NO: 44).

FD12 refers to the N-terminal strand 1 protein fused directly to strand 2. The nucleotide sequence encoding FD12 was created by the PCR-splicing overlap extension (SOE) technique using primers 12-1 ( SEQ ID NO: 45), 12-3 (SEQ ID NO: 46) and 12-2-1 (SEQ ID NO: 47).

FELD1-10aa and FELD1-15aa refer to N-terminal 2 stranded proteins fused by a 10 (GGGGS) 2 or 15 (GGGGS) 3 amino acid spacer, respectively, with the C-terminal strand 1. The plasmid containing the nucleotide sequence encoding FELD1 was used as a template to create the 10 or 15 amino acid spacers by reverse mutagenesis-PCR (IPCRM). For spacer 1-15, two primers were used (primer 1-1 Oaa, SEQ ID NO: 48 and primer 2-5aa, SEQ ID NO: 49). For spacer 1-10, two primers were used (primer 1-5aa, SEQ ID NO: 50 and primer 2-5aa). The resulting PCR fragment was annealed by ligation. It is resistant to Dpn I digestion, which only recognizes the sequence containing methylated adenine, while the model plasmid was digested by Dpnl. FELD1-10aa and FELD1-15aa refer to N-terminal 2-stranded proteins fused by a 10 (GGGGS) 2 or 15 (GGGGS) 3 amino acid spacer, respectively, with the C-terminal 2 strand. Fusion of FD12-10aa and FD12-15aa was produced similarly to that described above. For the 15 amino acid spacer, primer FD2-10aa (SEQ ID NO: 51) and primer FD1-5 aa (SEQ ID NO: 52) were used. For the 10 amino acid spacer, primer FD1-5aa and primer FD2-5 aa (SEQ ID NO: 53) were used. EXAMPLE 4

Bacterial expression and purification of Fel d1 fusion proteins with His-tea Nucleotide sequences encoding various Fel d1 fusion constructs as described in EXAMPLE 3 were subcloned into a pET-42T (+) expression system. T7 base, modified from plasmid pET-42a (+) (Novagen). The C-terminal of the fusion constructs was fused to a His-tag sequence, followed by GGC, a cysteine-containing amino acid linker as the second attachment site. The resulting constructs are named accordingly by adding "-HC" at their end.

FELD1-HC, FELD1-10aa-HC and FELD1-15aa-HC and FD12-15aa-HC were expressed and purified as follows: The plasmid was transformed into BL21 (DE3). Expression was induced by adding 1 mM IPTG to the OD600 culture of approximately. The culture was grown for an additional 30 hours at 20 s, collected and lysed by sonication in native lysis buffer (50 mM NaH 2 PO 4, 300 mM NaCl, 10 mM imidazole, pH 8.0).

The clarified bacterial lysate was brought to 50 ml with native lysis buffer. 5 ml of nickel nitrile tri-acetic acid (Ni-NTA) agarose (Qiagen) was added and the lysate was incubated, inverted, for one hour at 4 ° C. Nonspecifically bound proteins were removed by washing four times in native lysis buffer. Bound protein was eluted by resuspension of Ni-NTA agarose in 2 ml elution buffer (50 mM NaH 2 PO 4, 300 mM NaCl, 250 mM imidazole, pH 8.0).

EXAMPLE 5

Oxidative folded disulfide bonds in Fel d1 fusion proteins

Ni2 + affinity purified FELD1-HC consists of a variety of species with mixed disulfide bridges from 15 kD to 20 kD. The natural folding of FELD1-HC was obtained by repeated intramolecular mixing of the oxidized glutathione (GSSG, Appli-chem) and reduced glutathione (GSH, Applichem) disulfide bonds at a 1: 1 molar ratio. The reaction occurred for 24 hours immediately after elution of FELD1 in the elution buffer by the addition of 2.5 mM GSSG and 2.5 mM GSH at room temperature. Refolded FELD1-HC exhibited a single band at a molecular weight of 15 kD under non-reducing conditions (FIG. 1, the first panel, lane 2). FELD1-HC free potential sulfhydryl groups were alkylated using iodoacetamide (Sigma). Probes were treated with 5 mM iodoacetamide in 20 mM ammonium bicarbonate at pH 8.0 for 15 minutes at room temperature.

Refolded FELD1-HC was further purified to homogeneity by size exclusion chromatography (SEC) (Superdex 75 pg, Amersham Pharmacia Biosciences), equilibrated in PBS.

FELD1-10aa-HC and FELD1-15aa-HC were renatured by a process fundamentally the same as described above (FIG. 1, the two middle panels).

The natural folding of FD12-15aa-HC was also obtained by repeated mixing of disulfide bonds with oxidized glutation and reduced glutation in a 10: 1 molar ratio. After elution, FD12-15aa was diluted twenty-fold in FD12-15aa refolding buffer (50 mM Tris-CI, pH 8.5, 240 mM NaCl, 10 mM KCI, 1 mM EDTA, 3.550 PEG 0.05%, 0.1 mM GSH, 0.1 mM GSSH) and incubated at 4 ° C for 24 hours. Refolded FD12-15aa had a single ribbon at a molecular weight of 20 kD compared to the reduced shape running at 23 kD (FIG. 1, last panel). EXAMPLE 6

Fel d1 fusion proteins are recognized by epitope-specific monoclonal antibodies. The binding of Fel d1 compared to natural Fel f1 fusion proteins (nFel d1) with epitope-specific monoclonal antibodies (mAB) was determined by a sandwich ELISA using the deFel d1 (6F9 / 3E4) ELISA kit. Indoor biotechnologies (Cardiff, UK).

Briefly, anti-Fel d1 mAB 6F9, supplied as a 2 mg / ml stock solution, was diluted to 1: 1000 in 50 mM carbone-to-bicarbonate buffer, pH 9.6. Microtiter wells were coated with 100 µl of mAB 6F9 diluted per well in 4e overnight. The plates were washed three times with PBS-0.05% Tween20 (PBS-T) and then blocked with 100 µl blocking buffer (1% BSA (Sigma) in PBS-T). Microtiter wells were incubated for one hour with 100 µl of standard Fel d1 or nFel d1 fusion proteins (Indoors technologies;

United Kingdom) using double dilutions of 80-0.16 ng / ml. Reference nFel d1 was sub-standardized from CBER cat dandruff reference E10, which contains 13.47 U / ml Fel d1 (1 unit = 4 mg protein).

The plates were then washed and 100 µl diluted (1: 1000 in BSA / PBS-T to 1) of biotinyl containing anti-Fel d1 mAB 3E4 antibody were added and incubated for 1 hour at room temperature. The plates were washed three times with PBS-T using 100 µl diluted (1: 1000 in 1% BSA / PBS-T) Streptavidin Peroxidase (Sigma S5512, 0.25 mg reconstituted in 1 ml distilled water). After 30 minutes of incubation at room temperature, the wells were washed three times with PBS-T. Detection was performed with OPD substrate solution and 5% H2SO4 as stop solution. Absorbance was determined using an ELISA reading apparatus (BioRad) at 450 nm and to calculate arithmetic means and standard error of means (SEM), the EXCEL program (MS Office; Microsoft) was used. The results are presented in TABLE 1. Thus, the recognition of Fel d1 and nFel d1 fusion proteins by epitope-specific mABs reflects the high antigenicity similarity of both proteins.

TABLE 1

<table> table see original document page 54 </column> </row> <table> EXAMPLE 7

Coupling of Fel d1 fusion proteins to QB-derived VLPs

A 143 æM solution of VLP QB in HEPES buffer (20 mM HEPES, 150 mM NaCl, pH 7.2) reacted with 5 times the molar excess (715 æM) of SMPH (Pierce) for 30 minutes at 25 eC, under agitation. SMPH was taken from a 50 mM stock dissolved in dimethyl sulfoxide. The reaction products were dialyzed for two PBS changes using a dialysis unit with a molecular weight cut-off of 10,000 Da (Slide-A-Lyzer, Pierce. Dialysis was conducted at 4 ° C at room temperature). , with a buffer greater than> 1000 times that of the reaction mixture.

Prior to coupling SMPH-depleted Fel d1 to NPVB fusion proteins, FELD1, FELDMOaa, FELD1-15aa and FD12-15aa, respectively, as obtained according to EXAMPLE 5, were incubated with TCEP (Pierce, Perbio Science) in equimolar quantities for 30 minutes at room temperature.

Fel d1 fusion proteins were added in molar surplus 5 times greater than that of the 143 µM solution of QPH NPV-derived NPVs. The reaction volume was 650 ul and multiple reactions were performed in parallel. Reactions were incubated for 4 hours at room temperature under shaking. After coupling, aliquots were centrifuged at 16,000 x g for 3 minutes at 4 ° C until transformed into pellet insoluble material. The supernatants were pooled in new tubes. Coupling of Fel d1 fusion proteins to QB VLP was evaluated by reducing SDS-PAGE.

Coupling of Fel d1 fusion proteins to QB reassembled VLP (obtained from EXAMPLE 1) occurs in substantially the same manner as described above.

EXAMPLE 8

FELD1 coupling. FELD1-15aa and FD12-15aa to HBcAa1-185-Lis

The HBcAg1-185-Lys construct, its expression and purification were substantially described in EXAMPLE 2-5 of WO 03/040164. HBcAg1-185-Lis VLP Solution 120 µM VLP in 20 mM Hepes, 150 mM NaCl, pH 7.2, reacts for 30 minutes with a 25-fold larger molar excess of SMPH (Pierce) diluted from stock solution in DMSO at 25 ° C on a rotary shaker. The reaction solution is then twice dialyzed twice for two hours against 1 liter of 20 mM Hepes, 150 mM NaCl, pH 7.2, at 4 ° C. The dialyzed mixture of HBcAg1-185-Lys then reacts with the recombinant Fel d1 obtained in EXAMPLE 5. In the coupling reaction, FELD1, FELD1-15aa to FD12-15aa, respectively / are present in a twice molar excess. greater than that of the derivatized HB-10 cAg1-185-Lis VLP. The coupling reaction proceeds for four hours at 25 ° C on a rotary shaker.

EXAMPLE 9

FELD1 coupling. FELD1-15aa and FD12-15aa with AP205-derived VLPs

The preparation of AP205 VLP VLP has been described in EXAMPLE 1 and 2 in WO 2004/007538. The VLP derivatization of AP205 is substantially the same as that described in EXAMPLE 7 for VLP Qp. Prior to coupling the SMPH derivatized Fel d1 to NPV AP205 fusion proteins, the FELD1, FELD1-10aa, FELD1-15aa and FD12-15aa, respectively, as obtained according to EXAMPLE 5, were incubated with TCEP (Pierce, Perbio Science) in equimolar quantities for 30 minutes at room temperature.

Fel d1 fusion proteins are added in molar excess 5 times greater than that of the 143 µM solution of SMPH-derivatized AP205 VLPs. Reactions were incubated for 4 hours at room temperature under shaking.

Coupling of Fel d1 fusion proteins with reassembled VLP of AP205 (obtained from EXAMPLE 2) occurs in substantially the same manner as described above.

EXAMPLE 10

Production of the bacteriophage-based composition of the invention QB.

A 601 E. coli AB259 culture (5x10 7 cells / ml) was grown for 2-3 hours at 37 ° C under intense aeration (1 volume of air / volume of minute in culture) to obtain a culture of approximately 2-4x109 cells / ml. Clarified Qp phage lysate was inoculated into an infection multiple of 5 and CaCl 2 was added to a final concentration of 2.2 mM. After a 5 minute adsorption phase, aeration was intensified (1.5 volumes air / minute volume in culture). The cells were cultured for a further 3 hours until OD65 nm reached equilibrium, producing 4-6x10 12 phage particles in the culture. These were purified as follows: E.coli was lysed by the addition of 10 ml CHCl3 / I culture, 0.1 mg lysozyme / I culture and EDTA to a final concentration of 20 mM. The lysate was clarified by centrifugation in a continuous cooled centrifuge and phage particles sedimented from the lysate by ammonium sulfate precipitation (500 g / l, yielding approximately 66% saturation). The suspension was initially decanted and the precipitate isolated by centrifugation for 30 minutes using a W.R. rotor Janetzki K26 centrifuge. 6 x 500 ml at 6000 rpm, or in a Beckman J21 C centrifuge with JA-10 rotor. The pellet was again solubilized in NT buffer (0.15 NaCl and 0.1% Trypton) and clarified by centrifugation. The supernatant was precipitated with ammonium sulfate (500 g / l, approximately 66% saturation added). The precipitate was isolated by centrifugation, resolubilized in NT buffer and clarified again by centrifugation. Phage particles were isolated from the resulting supernatant by 3.5 h ultra-centrifugation using a Beckman type 35 rupture at 32,000 rpm. Pelleted phages were resuspended in NT buffer and purified by ultracentrifugation on a continuous conventional CsCI gradient. Centrifugation was conducted using a Beckman Ti-70 rotor (8x38.5) rotor at 55,000 rpm for 20 hours. The phage particles were subsequently dialyzed into 20 mM Hepes, 150 mM NaCl, pH 7.4 for use in chemical cross-linking steps as described in EXAMPLE 7.

EXAMPLE 11

Preparation of the GA-based composition of the invention

A 12 I E. coli Q 13 Hfr RNASse I 'culture in M9 medium containing 2% casein hydrolyzate, 0.5% yeast extract, 0.2% glucose was grown to an OD540 nm of 0, 6-0.7, which corresponds to approximately 2x108 cells / ml, and infected with GA phage in an infection multiple of 10-20. The culture grew for an additional 2.5 to 3 hours at 37 ° C and produced approximately 1011 phage particles throughout the culture. The cells were lysed by the addition of 1-2% v / v CHCl3 and the culture incubated for 15 minutes. The lysate was clarified by centrifugation for 30 minutes at 5000 rpm in a Janetzi K26 rotor. The particles were indeed precipitated with ammonium sulfate (60% saturation) from the culture medium for several days at 4 ° C. The suspension was initially decanted, and the precipitate isolated by centrifugation for 30 minutes at 6000 rpm on a Janetzki K26 rupture. The pellet was resuspended in NET buffer (20 mM Tris, pH 7.8, 150 mM NaCl, 5 mM EDTA), and the particles extracted by various centrifugation and resuspension cycles in small portions of the NET buffer. The capsid containing parts were pooled and precipitated with 60% ammonium sulfate. Resuspended particles in NET buffer were purified three times on a Sepharose 4B column and subsequently on two sucrose gradients. Briefly, a 7 ml sucrose gradient with the following v / v, 50%, 43%, 36%, 29% and 22% concentrations was prepared in NET buffer in centrifuge tubes. The phage solution (in NET buffer) was layered on gradient and centrifuged for 17 hours in a Beckman SW 28 rotor at 25,000 rpm. The capsid containing fractions were pooled and separated from sucrose by gel filtration on a Sepharose 4B column. The phage particles were subsequently dialyzed in water and lyophilized for later use.

The condition of coupling of Fel d1 fusion proteins with the bacteriophage Qp or GA is substantially the same as the condition of coupling with Qp VLP as set forth in EXAMPLE 7.

EXAMPLE 12

Fel d1 Qp fusion proteins are highly immunogenic in mice BALB / c mice were immunized sc with 50 µg of Qp-FELD1 obtained from EXAMPLE 7 or 50 µg of Qp mixed with recombinant Fel d1 (obtained from EXAMPLE 5 ) on days 0, 14 and 21 and blood samples were collected on days 0, 21 and 28. Fel d1 specific antibodies were determined by ELISA using FELD1 for coating (10 µg / ml). Briefly, 96 F96 wells were used, which were pre-coated at 4 ° C overnight with 10 µg / ml FELD1 in 0.1 M NaHCO 3 and pH 9.6. The plates were washed four times with PBS-Tween 20 and the background was reduced by incubating the plates 2 hours at 37 ° C with Stop Buffer (2% BSA, Sigma) in PBS-Tween20. Serum was diluted in serum dilution buffer (2% BSA, 1% FCS in PBS-Tween20). Two dilution and incubation steps were conducted for 2 hours at room temperature on an ELISA plate shaker. The plates were washed five times and detection antibody, diluted 1: 1000 (Sigma-coupled anti-mouse HRG IgG), was incubated for 1 hour at room temperature. The plates were washed four times with PBS-Tween20 and detection was performed using OPD substrate solution (0.066 M Na2HP04, 0.035 citric acid, pH 5.0, containing 10 mg OPD (Fluka) and 8 Ml H2 O at 30% (Fluka) per 25ml) and 5% H 2 SO 4 in H 2 O as a stop solution. Absorbance was determined using a 450 nm ELISA reader and for calculation of arithmetic means and standard error of means (SEM) the EXCEL program (Microsoft) was used.

Mice immunized with Qp-FELD1 described a maximum half-absorption of 120,000 and 75,000 on day 21 and 289, respectively. On the other hand, mice immunized with a non-ligated Qp / FELD 1 mixture showed lower titers of 7000 and 6000 at 219 and 28e day, respectively.

EXAMPLE 13

Aluminum mouse immunization as adjuvant 7-8 week Balb / c mice were vaccinated three times (days 0, 14 and 28) with 50 µg of prior art QpVLP-FELD1-HC. Vaccines were diluted in 200 µl sterile PBS or 100 µl PBS and 100 µl AluGel-S (Serva), respectively, and injected subcutaneously into the left and right inguinal region.

Serum was collected on days 14, 21, 28, 42, 56, 84 and 112. Specific antibodies against natural Fel d1, FELD-HC and QB VLP were determined by ELISA. Microtiter plates were coated overnight with 1

ng / ml natural Fel d1 (nFel d1, Indoors biotechnologies), 10 mg / ml FELD1-HC and 10 mg / ml Qb VLP, respectively. After washing (Twe-en 20 / 0.05% PBS) and quenching with 2% BSA in PBS, serum was added at different dilutions in 2% BSA / 1% FCS / PBS.

Subsequently, the ELISA was conducted by the standard method. Q vaccines (3 VLP-FELD1-HC induced a high lasting Feldl specific antibody response to natural FELD1 and FELD1. Antibody titer was higher in the presence of aluminum than in the absence of α-lumenium (TABLE 2) .

TABLE 2

<table> table see original document page 60 </column> </row> <table>

EXAMPLE 14

QB-coupled FELD1 dramatically reduced in vitro allergic potential

To test Qp-FELD1-HC's ability to trigger an in vitro allergic reaction, basophils were isolated from the blood of three allergic donor cats. In an allergic cat, these basophils are coated with Fel d1-specific IgE and respond with regulated CD63 increase to allergen stimulation. Thus, basophils from the allergic individual were stimulated with graded amounts of Qp-FELDI-HC or FELD1-HC alone and the regulated CD63 increase was determined by flow cytometry. While FELD1-HC (obtained from EXAMPLE 5) elicited a strong regulated increase in CD63, at the lowest dilution tested (approximately 0.2ng / ml), QP-FELD1-HC (obtained from EXAMPLE 7) exhibited a potentially allergic drastically. reduced and required 100-1000 times higher amounts (approximately 70ng / ml) of FELD1-HC for basophils to respond.

This test was also repeated for FELD-15aa-HC and FD12-15aa-HC fusion proteins, isolated or coupled with Qp. The results are shown in FIG 2. These Fel d1 fusion proteins, when coupled with Qp, all exhibit drastically reduced allergic potential.

EXAMPLE 15

Qp-coupled FELD1 cannot trigger a skin puncture reaction in an allergic individual

If allergens are introduced by a puncture of the skin of an allergic individual, a local edema will be created in approximately 20 minutes due to activation of mast cells in the skin. In this example, the skin test was employed to determine the allergic potential of Qp-FELD1-HC (obtained from EXAMPLE 7). Graded amounts of Qp-FELD1-HC or corresponding amounts of FELD1-HC (obtained from E-XEMPLO 5) were introduced into the skin of an allergic cat and the skin reaction was evaluated 20 minutes later. Qp-FELD1-HC was not able to trigger a skin reaction while FELD1-HC was active at concentrations over 100 times lower (TABLE 3).

TABLE 3: Corresponding amounts of FELD1-HC present in both preparations.

<table> table see original document page 61 </column> </row> <table> EXAMPLE 16

Immunization of an allergic individual with QB-FELD1-HC reduces skin prick test reactivity

In order to test Qp-FELD-HC's ability to alleviate clinical symptoms, a patient suffering from cat allergy was vaccinated with 17ug Qp-FELDI-HC (obtained from EXAMPLE 7) on day 0 (3 injections of 2, 5 and 10 æg), 40 æg on day 7 (3 injections of 10, 10 and 20 æg) and 50 æg on day 14 (2 injections of 10 and 40 æg). Skin puncture tests were performed with a standardized cat extract on days 0, 14, and 21, and the central swollen reaction diameters were quantified (FIG 3). At 3 weeks, allergen concentrations 1000 times higher were required to induce clinical symptoms in the skin puncture test than the initial amount needed to induce symptoms in the puncture test. EXAMPLE 17 Immunization of an allergic individual with QB-FELD1-HC reduces nasal symptoms on nasal challenge test

In order to test Qp-FELD1-HC's ability to alleviate clinical symptoms, a cat allergy patient was vaccinated with 17ug Qp-FELDI-HC (obtained from EXAMPLE 7) on day 0 (3 injections of 2, 5 and 10 µg), 40 µg on day 7 (3 injections of 10, 10 and 20 µg) and 50 µg on day 14 (2 injections of 10 and 40 µg). Nasal challenge tests were performed with a standardized cat extract on days 0, 14 and 21 and clinical symptoms were assessed at each dose escalation level (FIG 4A) and an overall allergic score was prepared according to (FIG 4B). . At 3 weeks, 100 to 1000 higher amounts of allergen were required to induce clinical symptoms and the overall allergic score was greatly reduced.

EXAMPLE 18

Packaging of immunostimulatory nucleic acids in QB VLP

Disassembled and purified Qp coat protein was obtained as described in EXAMPLE 1 (A) and (B). Reassembly: p-mercaptoethanol was added to the 10 ml fraction of the dimer to a final concentration of 10%, and 300 µl of an oligodeoxy nucleotide (CpG) 2 OOA solution containing 12.3 nmols of oligonucleotide was added. The reassembly mixture was initially dialyzed 30 ml of NET buffer (20 nM Tris-HCl, pH 7.8 with 0.5 mM EDTA and 150 mM NaCl) containing 10% beta-mercaptoethanol for 2 hours at room temperature. 4 ° C and then dialyzed continuously with an 8 ml / h NET buffer flow for 4 days at 4 ° C. The reassembly mixture was then desalted in water by dialysis with 6 buffer changes (4 X 100 ml, 2 X 1 liter).

Coupling of Fel d1 fusion proteins with QpVLP containing CpG within is conducted in substantially the same manner as described in EXAMPLE 7.

Example 19

Fel d1-specific serum inhibited Fel d1 fusion protein-induced basophil degranulation in vitro

In order to test the ability of anti-Fel d1 specific IgG to inhibit degranulation, basophils from an allergic individual were stimulated with a defined amount of FELD1-HC or FELD1-15aa-HC, preincubated with a serial dilution of amounts. decreases in IgGs isolated from Qp-FELD1-HC immunized mice. CD63 regulated increase was determined by flow cytometry. Anti-Fel d1 IgG blocked Fel d1-induced degranulation at all concentrations tested, while control IgG showed no effect (TABLE 4).

TABLE 4

<table> table see original document page 63 </column> </row> <table> <table> table see original document page 64 </column> </row> <table>

EXAMPLE 20

Immunization of Fel d1 Allergic Mice with Q (3-FELD1-HC

An experimental asthma model of allergic airway inflammation in mice was used to evaluate the effects of vaccination against the natural allergen Fel d1 on IgE antibody response in BALB / c mice serum and LBA (Bronchoalveolar lavage). mice per group were sensitized by natural Fel d1 peritoneal notch in AlumGel-S on day 0. Mice were vaccinated subcutaneously on day 35 and 49 with 50ug Q (3 alone or 50ug Q (3-FELD1-HC , prior to two subsequent intranasal tests on day 63 and day 70. 5 days after the last intranasal test, mice were sacrificed to collect serum and LLBA (bronchoalveolar lavage fluid) to be analyzed for humoral immune response. (IgE subclass titers) by ELISA.

ELISA plates were coated with an anti-mouse IgE mAB (2ug / ml) diluted in carbonate buffer overnight at 4 ° C. After blocking the plates with 5% PBS / BSA for 2 hours, the plates were incubated for two more hours with serum (first well: 1: 100 prediluted, then 1: 3 dilution over 8 steps) or LBA (first well, pure LBA, then 1: 3 dilution over 8 steps) from sensitized, vaccinated and antigen tested mice. After 2 hours of sample incubation, serum IgE and BAL were detected with a mouse anti-mouse IgE-HRP labeled antibody prior to detection with OPD.64 substrate.

TABLE 5

<table> table see original document page 65 </column> </row> <table> Sequence Listing

<110> Cytos Biotechnology AG Bachmann, Martin F Monika, BAUER F Dietmeier, Klaus Schmitz, Nicole Uzinger, Stefan

<120> CAT ALLERGEN ASSEMBLIES AND USES OF THE SAME

<130> P1048PC00

<150> 60 / 662,918 <151> 2005-03-18

<160> 69

<170> Patentln version 3.2

<210> 1

<211> 132

<212> PRT

<213> Q-beta bacteriophage

<400> 1

Ala Lys Leu Glu Thr Go Thr Leu Gly Asn lie Gly Lys Asp Gly Lys 1 5 10 15

Gln Thr Read Go Go Read Asn Go Pro Arg Gly Go Asn Go Pro Asn Gly Go Go 20 25 30

Wing Be Read To Be Gln Wing Gly Wing Go To Wing Read Leu Glu Lys Arg Go 35 40 45

Thr Gonna Be Gonna Be Gln Pro Gonna Be Arg Asn Arg Lys Asn Tyr Lys Go 50 55 60

Gln Goes Lys Lie Gln Asn Pro Thr Cys Wing Thr Asn Gly Be Cys 65 70 75 80

Asp Pro Will Go Thr Arg Gln Wing Tyr Wing Asp Go Will Thr Phe Be Phe 85 90 95

Thr Gln Tyr Be Thr Asp Glu Glu Arg Wing Phe Go Arg Thr Glu Leu 100 105 110

Ala Ala Leu Leu Ala Ser Pro Leu Leu lie Asp Ala lie Asp Gln Leu 115 120 125

Asn Pro Wing Tyr 130 <210> 2

<211> 329

<212> PRT

<213> Q-beta bacteriophage

<400> 2

Met Ala Lys Leu Glu Thr Go Thr Leu Gly Asn lie Gly Lys Asp Gly 1 5 10 15

Lys Gln Thr Read Go Read Asn Pro Arg Gly Go Read Asn Pro Thr Asn Gly 20 25 30

Go Wing Be Read Be Gln Wing Gly Wing Go Go Wing Read Glu Lys Arg 35 40 45

Will Thr Will Be Will Be Gln Pro Be Arg Asn Arg Lys Asn Tyr Lys 50 55 60

Go Gln Go Lys lie Gln Asn Pro Thr Wing Cys Thr Wing Asn Gly Ser 65 70 75 80

Cys Asp Pro Will Be Thr Thr Gln Wing Tyr Wing Asp Will Thr Thr Phe 85 90 95

Phe Thr Gln Tyr Be Thr Asp Glu Glu Arg Wing Phe Go Arg Thr Glu 100 105 110

Leu Ala Ala Leu Leu Ala Ser Pro Leu Leu lie Asp Ala lie Asp Gln 115 120 125

Leu Asn Pro Wing Tyr Trp Thr Leu Leu lie Wing Gly Gly Gly Ser Gly 130 135 140

Ser Lys Pro Asp Pro Will Lie Pro Asp Pro Pro Lie Asp Pro Pro 145 150 155 160

Gly Thr Gly Lys Tyr Thr Cys Pro Phe Ala lie Trp Ser Leu Glu Glu 165 170 175

Go Tyr Glu Pro Pro Lys Asn Arg Pro Trp Pro lie Tyr Asn Wing 180 185 190

Go Glu Leu Gln Pro Arg Glu Phe Asp Go Wing Leu Lys Asp Leu Leu 195 200 205

Gly Asn Thr Lys Trp Arg Asp Trp Asp Be Arg Read Le Tyr Thr Thr 210 215 220Phe Arg Gly Cys Arg Gly Asn Le Gly Tyr lie Asp Leu Asp Wing Thr Tyr 225 230 235 240

Leu Wing Thr Asp Gln Ala Met Arg Asp Gln Lys Tyr Asp lie Arg Glu 245 250 255

Gly Lys Lys Pro Gly Phe Gly Ashe Gly Asn lie Glu Arg Phe lie Tyr Leu 260 265 270

Lys Ser lie Asn Ala Tyr Cys Ser Leu Ser Asp lie Ala Tyr His 275 280 285

Wing Asp Gly Go lie Go Gly Phe Trp Arg Asp Pro To Be Gly Gly 290 295 300

Ala lie Pro Phe Asp Phe Thr Lys Phe Asp Lys Thr Lys Cys Pro lie 305 310 315 320

Gln Wing Go lie Go Go to Arg Wing 325

<210> 3

<211> 129

<212> PRT

<213> Bacteriophage R17

<400> 3

Ala Ser Asn Phe Thr Gln Phe Will Leu Go Asn Asp Gly Gly Thr Gly 1 5 10 15

Asn Go Thr Go Ala Pro Be Asn Phe Ala Asn Gly Go Ala Glu Trp 20 25 30

lie Be Ser Asn Ser Arg Be Gln Wing Tyr Lys Go Thr Cys Be Go 35 40 45

Arg Gln Ser Be Ala Gln Asn Arg Lys Tyr Thr lie Lys Go Glu Go 50 55 60

Pro Go Lys Go Wing Gln Thr Go Gly Gly Go Glu Leu Go Go Wing 65 70 75 80

Wing Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr lie Pro lie Phe Ala 85 90 95

Thr Asn Be Asp Cys Glu Leu lie Go Lys Wing Met Gln Gly Leu Leu 100 105 110Lys Asp Gly Asn Pro Ile Pro Be Wing Ile Wing Asn Wing Gn Ile 115 120 125

Tyr

<210> 4

<211> 130

<212> PRT

<213> Bacteriophage fr

<400> 4

Met Ala Ser Asn Phe Glu Glu Phe Will Read Leu Go Asp Asn Gly Gly Thr 15 10 15

Gly Asp Goes Lys Goes Wing Pro Be Asn Phe Asn Wing Gly Goes Wing Glu 20 25 30

Trp Ile Be Ser Asn Ser Arg Be Gln Wing Tyr Lys Will Thr Cys Be 35 40 45

Go Arg Gln Be Ser Asa Asn Arg Lys Tyr Thr Go Lys Go Glu 50 55 60

Go Pro Lys Go Wing Thr Gln Go Gln Gly Gly Go Glu Leu Pro Go 65 70 75 80

Wing Wing Trp Arg Be Tyr Met Asn Met Glu Read Thr Ile Pro Go Phe 85 90 95

Wing Thr Asn Asp Asp Cys Wing Leu Ile Goes Lys Wing Leu Gln Gly Thr 100 105 110

Phe Lys Thr Gly Asn Pro Ile Wing Thr Wing Ile Wing Wing Asn Ser Gly 115 120 125

Ile Tyr 130

<210> 5

<211> 130

<212> PRT

<213> Bacteriophage GA

<400> 5

Met Wing Thr Read Le Arg Be Phe Go Read Le Go Go Asp Gly Gly Thr Gly 15 10 15Asn Go Thr Go Go Pro Go Be Asn Wing Asn Gly Go Ala Glu Trp 20 25 30

Leu Be Asn Asn Be Arg Be Gln Wing Tyr Arg Go Thr Wing Be Tyr 35 40 45

Arg Wing Be Gly Wing Asp Lys Arg Lys Tyr Wing Lie Lys Leu Glu Go 50 55 60

Pro Lys lie Go Thr Gln Go Go Asn Gly Go Glu Leu Pro Gly Ser 65 70 75 80

Wing Trp Lys Wing Tyr Wing Ser lie Asp Leu Thr lie Pro lie Phe Wing 85 90 95

Wing Thr Asp Asp Go Thr Go lie Ser Lys Ser Leu Wing Gly Leu Phe 100 105 110

Lys Goes Gly Asn Pro lie Ala Glu Ala lie Ser Ser Gln Ser Gly Phe 115 120 125

Tyr Wing 130

<210> 6 <211> 132 <212> PRT

<213> Bacteriophage SP

<400> 6

Met Ala Lys Read Asn Gln Go Thr Read Be Lys lie Gly Lys Asn Gly 15 10 15

Asp Gln Thr Read Thr Read Read Thr Pro Arg Gly Goes Asn Pro Thr Asn Gly 20 25 30

Go Wing Be Leu Be Glu Wing Gly Wing Go To Wing Leu Glu Lys Arg 35 40 45

Go Thr Go Go Go Wing Gln Pro Be Arg Asn Arg Lys Asn Phe Lys 50 55 60

Go Gln lie Lys Leu Gln Asn Pro Thr Cys Wing Thr Asp Cys Wing 65 70 75 80

Asp Pro Will Be Thr Thr Be Ala Phe Wing Asp Will Thr Thr Be Leu Phe 85 90 95Thr Be Tyr Be Thr Asp Glu Glu Arg Wing Leu lie Arg Thr Glu Leu 100 105 110

Ala Ala Leu Leu Ala Asp Pro.Leu lie Go Asp Ala lie Asp Asn Leu 115 120 125

Asn Pro Wing Tyr 130

<210> 7

<211> 329

<212> PRT

<213> Bacteriophage SP

<400> 7

Ala Lys Read Asn Gln Go Thr Read Be Lys lie Gly Lys Asn Gly Asp 1 5 10 15

Gln Thr Leu Thr Leu Thr Pro Arg Gly Goes Asn Pro Thr Asn Gly Goes 20 25 30

Wing Be Leu Be Glu Wing Gly Wing Go To Wing Leu Glu Lys Arg Go 35 40 45

Thr Will Be Go Ala Gln Pro Be Arg Asn Arg Lys Asn Phe Lys Go 50 55 60

Gln lie Lys Leu Gln Asn Pro Thr Cys Wing Thr Arg Asp Wing Cys Asp 65 70 75 80

Pro Will Go Thr Arg Be Wing Phe Wing Asp Will Thr Read Be Phe Thr 85 90 95

Ser Tyr Ser Thr Asp Glu Glu Arg Wing Leu lie Arg Thr Glu Leu Wing 100 105 110

Wing Leu Leu Wing Asp Pro Leu lie Go Asp Wing Ala Asp Asn Leu Asn 115 120 125

Pro Wing Tyr Trp Wing Wing Read Leu Go Wing Be Ser Gly Gly Gly Asp 130 135 140

Asn Pro Be Asp Pro Asp Go Pro Go Go Pro Asp Go Lys Pro Pro 145 150 155 160

Asp Gly Thr Gly Arg Tyr Lys Cys Pro Phe Ala Cys Tyr Arg Leu Gly 165 170 175Ser lie Tyr Glu Go Gly Lys Glu Gly Ser Asp lie Tyr Glu Arg 180 185 190

Gly Asp Glu Will Be Go Thr Thr Phe Asp Tyr Wing Leu Glu Asp Phe Leu 195 200 205

Gly Asn Thr Asn Trp Arg Asn Trp Asp Gln Arg Read Asp Tyr Asp 210 215 220

lie Ala Asn Arg Arg Arg Cys Arg Gly Asn Gly Tyr lie Asp Leu Asp 225 230 235 240

Wing Thr Wing Met Gln Be Asp Asp Phe Will Read Gly Arg Tyr Gly 245 250 255

Go Arg Lys Go Lys Phe Pro Gly Wing Phe Gly Ser lie Lys Tyr Leu 260 265 270

Leu Asn lie Gln Gly Asp Wing Trp Leu Asp Leu Be Glu Go Thr Wing 275 280 285

Tyr Arg Be Tyr Gly Met Will Lie Gly Phe Trp Thr Asp Be Lys Ser 290 295 300

Pro Gln Read Pro Thr Asp Phe Thr Gln Phe Asn Ser Wing Asn Cys Pro 305 310 315 320

Go Gln Thr Go lie lie lie Pro Ser 325

<210> 8

<211> 130

<212> PRT

<213> Bacteriophage MS2

<400> 8

Met Ala Be Asn Phe Thr Gln Phe Go Read Le Go Go Asp Gly Gly Thr 1 5 10 15

Gly Asp Go Thr Go Wing Pro Be Asn Phe Asn Wing Gly Go Wing Glu 20 25 30

Trp lie Be Ser Asn Ser Arg Be Gln Wing Tyr Lys Will Thr Cys Be 35 40 45

Go Arg Gln Go Be Wing Gln Asn Arg Lys Tyr Thr lie Lys Go Glu 50 55 60Go Pro Lys Go Wing Thr Gln Thr Go Gly Gly Go Glu Leu Pro Go 65 70 75 80

Wing Wing Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr lie Pro lie Phe 85 90 95

Wing Thr Asn Be Asp Cys Glu Leu Lie Go Lys Wing Met Gln Gly Leu 100 105 110

Leu Lys Asp Gly Asn Pro lie Pro Ser Ala lie Ala Wing Asn Ser Gly 115 120 125

lie Tyr 130

<210> 9

<211> 133

<212> PRT

<213> Bacteriophage M11

<400> 9

Met Ala Lys Leu Gln Ala lie Thr Leu Ser Gly lie Gly Lys Lys Gly 1 5 10 15

Asp Go Thr Read Asp Read Asn Pro Arg Gly Go Asn Pro Thr Asn Gly 20 25 30

Go Wing Wing Leu Be Glu Wing Gly Wing Go To Wing Leu Glu Lys Arg 35 40 45

Will Thr lie Will Be Gln Pro Be Arg Asn Arg Lys Asn Tyr Lys 50 55 60

Go Gln Go Lys lie Gln Asn Pro Thr Be Cys Thr Wing Be Gly Thr 65 70 75 80

Cys Asp Pro Will Be Thr Thr Be Wing Tyr Be Asp Will Thr Thr Be 85 90 95

Phe Thr Gln Tyr Be Thr Go Glu Glu Arg Wing Wing Leu Go Arg Thr Glu 100 105 110

Leu Gln Ala Leu Leu Ala Asp Pro Met Leu Go Asn Ala lie Asp Asn 115 120 125

Read Asn Pro Wing Tyr 130 <210> 10

<211> 133

<212> PRT

<213> Bacteriophage MX1

<400> 10

Met Ala Lys Leu Gln Ala lie Thr Leu Ser Gly lie Gly Lys Asn Gly 1 5 10 15

Asp Go Thr Read Asn Read Asn Pro Arg Gly Go Asn Pro Thr Asn Gly 20 25 30

Go Wing Wing Leu Be Glu Wing Gly Wing Go To Wing Leu Glu Lys Arg 35 40 45

Will Thr lie Will Be Gln Pro Be Arg Asn Arg Lys Asn Tyr Lys 50 55 60

Go Gln Go Lys lie Gln Asn Pro Thr Be Cys Thr Wing Be Gly Thr 65 70 75 80

Cys Asp Pro Will Go Thr Arg Be Wing Tyr Wing Asp Go Thr Thr Phe 85 90 95

Phe Thr Gln Tyr Be Thr Asp Glu Glu Arg Wing Wing Read Go Arg Thr Glu 100 105 110

Leu Lys Ala Leu Leu Asp Pro Wing Met Leu lie Asp Ala lie Asp Asn 115 120 125

Read Asn Pro Wing Tyr 130

<210> 11 <211> 330 <212> PRT

<213> Bacteriophage NL95 <400> 11

Met Ala Lys Leu Asn Lys Go Thr Leu Thr Gly lie Gly Lys Ala Gly 15 10 15

Asn Gln Thr Read Thr Read Le Thr Pro Arg Gly Goes Asn Pro Thr Asn Gly 20 25 30

Go Ala Be Leu Be Glu Ala Gly Ala Go To Ala Leu Glu Lys Arg 35 40 45Go Thr Will Go Go Ala Gln Pro Be Arg Asn Arg Lys Asn Tyr Lys 50 55 60

Go Gln lie Lys Leu Gln Asn Pro Thr Cys Wing Thr Lys Asp Cys Wing 65 70 75 80

Asp Pro Will Be Thr Arg Be Gly Be Arg Asp Pro Will Be Thr Read Phe 85 90 95

Thr Be Tyr Be Thr Glu Arg Glu Arg Wing Leu lie Arg Thr Glu Leu 100 105 110

Ala Ala Leu Leu Lys Asp Asp Leu lie Go Asp Ala lie Asp Asn Leu 115 120 125

Asn Pro Wing Tyr Trp Wing Wing Read Leu Wing Wing Ser Pro Gly Gly Gly 130 135 140

Asn Asn Pro Tyr Gly Go Pro Asp Be Pro Asn Go Lys Pro Pro 145 150 155 160

Gly Gly Thr Gly Thr Tyr Arg Cys Pro Phe Wing Cys Tyr Arg Arg Gly 165 170 175

Glu Leu lie Thr Glu Wing Lys Asp Gly Wing Cys Wing Leu Tyr Wing Cys 180 185 190

Gly Ser Glu Wing Leu Goes Glu Phe Glu Tyr Wing Leu Glu Asp Phe Leu 195 200 205

Gly Asn Glu Phe Trp Asp Asn Trp Asp Gly Arg Read As Lys Tyr Asp 210 215 220

lie Glu Thr His Arg Arg Cys Arg Gly Asn Gly Tyr Go Asp Leu Asp 225 230 235 240

Wing Will Be Met Gln Be Asp Glu Tyr Will Read Gly Wing Tyr Asp 245 250 255

Go Go Lys Met Gln Pro Gly Thr Phe Asp Be Pro Arg Tyr Tyr 260 265 270

Leu His Leu Met Met Asp Gly lie Tyr Go Asp Leu Wing Glu Go Thr Wing 275 280 285

Tyr Arg Be Tyr Gly Met Go lie Gly Phe Trp Thr Asp Be Lys Be 290 295 300Pro Gln Read Pro Thr Asp Phe Thr Arg Phe Asn Arg His Asn Cys Pro 305 310 315 320

Go Gln Thr Go Ile Go Ile Pro Be Leu 325 330

<210> 12

<211> 129

<212> PRT

<213> Bacteriophage f2

<400> 12

Ala Ser Asn Phe Thr Gln Phe Will Leu Go Asn Asp Gly Gly Thr Gly 1 5 10 15

Asn Go Thr Go Ala Pro Be Asn Phe Ala Asn Gly Go Ala Glu Trp 20 25 30

Ile Be Ser Asn Be Arg Be Gln Wing Tyr Lys Go Thr Cys Be Go 35 40 45

Arg Gln Ser Be Ala Gln Asn Arg Lys Tyr Thr Ile Lys Go Glu Go 50 55 60

Pro Go Lys Go Wing Gln Thr Go Gly Gly Go Glu Leu Pro Go Wing 65 70 75 80

Wing Trp Arg Ser Tyr Leu Asn Leu Glu Leu Thr Ile Pro Ile Phe Wing 85 90 95

Thr Asn Be Asp Cys Glu Leu Ile Goes Lys Wing Met Gln Gly Leu Leu 100 105 110

Lys Asp Gly Asn Pro Ile Pro Be Wing Ile Wing Wing Asn Wing Gly Ile 115 120 125

Tyr

<210> 13

<211> 128

<212> PRT

<213> Bacteriophage PP7

<400> 13

Met Be Lys Thr Ile Go Read Be Go Gly Glu Wing Thr Arg Thr Leu 1 5 10 15Thr Glu lie Gln Be Thr Wing Asp Arg Gln lie Phe Glu Glu Lys Go 20 25 30

Gly Pro Leu Goes Gly Arg Leu Arg Leu Thr Wing To Be Leu Arg Gln Asn 35 40 45

Gly Wing Lys Thr Wing Tyr Arg Go Asn Leu Lys Leu Asp Gln Wing Asp 50 55 60

Go Go Asp Cys Be Thr Go Go Cys Gly Glu Leu Pro Lys Go Arg 65 70 75 80

Tyr Thr Gln Will Trp Be His Asp Will Thr Lie Will Ward Asn Ser Thr 85 90 95

Glu Wing Be Arg Lys Be Read Tyr Asp Read Thr Thr Lys Be Read Go Wing 100 105 110

Thr Be Gln Go Glu Asp Leu Go Go Asn Leu Go Pro Leu Gly Arg 115 120 125

<210> 14

<211> 131

<212> PRT

<213> Bacteriophage AP205

<400> 14

Met Wing Asn Lys Pro Met Wing Gln Pro Lie Thr Be Thr Wing Asn Lys Lie 1 5 10 15

Will Trp Be Asp Pro Thr Arg Read Be Thr Thr Phe Be Wing Be Read 20 25 30

Leu Arg Gln Arg Goes Lys Goes Gly lie Ala Glu Reads Asn Asn Goes To Be 35 40 45

Gly Gln Tyr Gonna Be Gonna Tyr Lys Arg Pro Wing Pro Lys Pro Glu Gly 50 55 60

Cys Wing Asp Wing Cys Will lie Met Pro Asn Glu Asn Gln Ser lie Arg 65 70 75 80

Thr Go lie Ser Gly Ser Wing Glu Asn Leu Wing Thr Leu Lys Wing Glu 85 90 95

Trp Glu Thr His Lys Arg Asn Go Asp Thr Read Phe Ala Ser Gly Asn 100 105 110Ala Gly Leu Gly Phe Leu Asp Pro Thr Ala Ala Lie Will Be Asp 115 120 125

Thr Thr Wing 130

<210> 15

<211> 132

<212> PRT

<213> Artificial sequence

<220>

<223> Mutant Q-beta 240 bacteriophage

<400> 15

Wing Lys Leu Glu Thr Go Thr Leu Gly Asn 1 5 10

15

Gln Thr Read Go Go Read Asn Go Pro Arg Gly Go Asn Go Pro Asn Gly Go Go 20 25 30

Wing Be Read To Be Gln Wing Gly Wing Go To Wing Read Leu Glu Lys Arg Go 35 40 45

Thr Gonna Be Gonna Be Gln Pro Gonna Be Arg Asn Arg Lys Asn Tyr Lys Go 50 55 60

Gln Goes Lys Lie Gln Asn Pro Thr Cys Wing Thr Asn Gly Be Cys 65 70 75 80

Asp Pro Will Go Thr Arg Gln Lys Tyr Wing Asp Go Will Thr Phe Be Phe 85 90 95

Thr Gln Tyr Be Thr Asp Glu Glu Arg Wing Phe Go Arg Thr Glu Leu 100 105 110

Ala Ala Leu Leu Ala Ser Pro Leu Leu lie Asp Ala lie Asp Gln Leu 115 120 125

Asn Pro Wing Tyr 130

<210> 16

<211> 132

<212> PRT

<213> Artificial sequence

<220>

<223> Mutant Q-beta 243 bacteriophage

<400> 16Ala Lys Leu Glu Thr Go Thr Leu Gly Lys lie Gly Lys Asp Gly Lys 15 10 15

Gln Thr Read Go Go Read Asn Go Pro Arg Gly Go Asn Go Pro Asn Gly Go Go 20 25 30

Wing Be Read To Be Gln Wing Gly Wing Go To Wing Read Leu Glu Lys Arg Go 35 40 45

Thr Gonna Be Gonna Be Gln Pro Gonna Be Arg Asn Arg Lys Asn Tyr Lys Go 50 55 60

Gln Goes Lys Lie Gln Asn Pro Thr Cys Wing Thr Asn Gly Be Cys 65 70 75 80

Asp Pro Will Go Thr Arg Gln Lys Tyr Wing Asp Go Will Thr Phe Be Phe 85 90 95

Thr Gln Tyr Be Thr Asp Glu Glu Arg Wing Phe Go Arg Thr Glu Leu 100 105 110

Ala Ala Leu Leu Ala Ser Pro Leu Leu lie Asp Ala lie Asp Gln Leu 115 120 125

Asn Pro Wing Tyr 130

<210> 17

<211> 132

<212> PRT

<213> Artificial sequence

<220>

<223> Mutant Q-beta 250 bacteriophage

<400> 17

Wing Arg Read Le Glu Thr Go Thr Leu Gly Asn lie Gly Arg Asp Gly Lys

1 5 10

Gln Thr Read Go Go Read Asn Go Pro Arg Gly Go Asn Go Pro Asn Gly Go Go

20 25 30

Wing Be Leu Be Gln Wing Gly Wing Go To Wing Leu Glu Lys Arg Go

35 40 45

Thr Will Be Gonna Be Gln Pro Be Arg Asn Arg Lys Asn Tyr Lys Go 50 55 60Gln Go Lys Lie Gln Asn Pro Thr Cys Wing Thr Asn Gly Be Cys Wing 65 70 75 80

Asp Pro Will Go Thr Arg Gln Lys Tyr Wing Asp Go Will Thr Phe Be Phe 85 90 95

Thr Gln Tyr Be Thr Asp Glu Glu Arg Wing Phe Go Arg Thr Glu Leu 100 105 110

Ala Ala Leu Leu Ala Ser Pro Leu Leu lie Asp Ala lie Asp Gln Leu 115 120 125

Asn Pro Wing Tyr 130

<210> 18

<211> 132

<212> PRT

<213> Artificial sequence

<220>

<223> Mutant A-beta 251 bacteriophage

<400> 18

Ala Lys Leu Glu Thr Go Thr Leu Gly Asn lie Gly Lys Asp Gly Arg 15 10 15

Gln Thr Read Go Go Read Asn Go Pro Arg Gly Go Asn Go Pro Asn Gly Go Go 20 25 30

Wing Be Read To Be Gln Wing Gly Wing Go To Wing Read Leu Glu Lys Arg Go 35 40 45

Thr Gonna Be Gonna Be Gln Pro Gonna Be Arg Asn Arg Lys Asn Tyr Lys Go 50 55 60

Gln Goes Lys Lie Gln Asn Pro Thr Cys Wing Thr Asn Gly Be Cys 65 70 75 80

Asp Pro Will Go Thr Arg Gln Lys Tyr Wing Asp Go Will Thr Phe Be Phe 85 90 95

Thr Gln Tyr Be Thr Asp Glu Glu Arg Wing Phe Go Arg Thr Glu Leu 100 105 110

Ala Ala Leu Leu Ala Ser Pro Leu Leu lie Asp Ala lie Asp Gln Leu 115 120 125

Asn Pro Wing Tyr130

<210> 19

<211> 132

<212> PRT

<213> Artificial sequence

<220>

<223> Mutant Q-beta 259 bacteriophage

<400> 19

Wing Arg Leu Glu Thr Go Thr Leu Gly Asn 1 5 10

15

Gln Thr Read Go Go Read Asn Go Pro Arg Gly Go Asn Go Pro Asn Gly Go Go 20 25 30

Wing Be Read To Be Gln Wing Gly Wing Go To Wing Read Leu Glu Lys Arg Go 35 40 45

Thr Gonna Be Gonna Be Gln Pro Gonna Be Arg Asn Arg Lys Asn Tyr Lys Go 50 55 60

Gln Goes Lys Lie Gln Asn Pro Thr Cys Wing Thr Asn Gly Be Cys 65 70 75 80

Asp Pro Will Go Thr Arg Gln Lys Tyr Wing Asp Go Will Thr Phe Be Phe 85 90 95

Thr Gln Tyr Be Thr Asp Glu Glu Arg Wing Phe Go Arg Thr Glu Leu 100 105 110

Ala Ala Leu Leu Ala Ser Pro Leu Leu lie Asp Ala lie Asp Gln Leu 115 120 125

Asn Pro Wing Tyr 130

<210> 20

<211> 185

<212> PRT

<213> Hepatitis B virus

<400> 20

Met Asp lie Asp Pro Tyr Lys Glu Phe Gly Wing Thr Go Glu Leu Leu 15 10 15

Be Phe Leu Pro Be Asp Phe Phe Pro Will Go Arg Asp Leu Read Asp 20 25 30Thr Wing Be Wing Leu Tyr Arg Glu Wing Leu Glu Be Pro Glu His Cys 35 40 45

Ser Pro His His Thr Ala Leu Arg Gln Ala lie Leu Cys Trp Gly Glu 50 55 60

Leu Met Thr Leu Wing Thr Trp Go Gly Asn Asn Leu Glu Asp Pro Wing 65 70 75 80

Be Arg Asp Leu Go Go Asn Tyr Go Asn Thr Asn Met Gly Leu Lys 85 90 95

lie Arg Gln Read Leu Trp Phe His lie Be Cys Leu Thr Phe Gly Arg 100 105 110

Glu Thr Gonna Read Glu Tyr Leu Gonna Be Phe Gly Gonna Trp Lie Arg Thr 115 120 125

Pro Pro Wing Tyr Arg Pro Pro Asn Wing Pro lie Leu Ser Thr Leu Pro 130 135 140

Glu Thr Thr Go Go Arg Arg Arg Asp Arg Gly Arg Be Pro Arg Arg 145 150 155 160

Arg Thr Pro Be Pro Arg Arg Arg Arg Be Gln Be Pro Arg Arg Arg 165 170 175

Arg Be Gln Be Arg Glu Be Gln Cys 180 185

<210> 21

<211> 188

<212> PRT

<213> hepatitis B virus

<400> 21

Met Asp lie Asp Pro Tyr Lys Glu Phe Gly Wing Thr Go Glu Leu Leu 1 5 10 15

Be Phe Leu Pro Be Asp Phe Phe Pro Be Go Arg Asp Leu Read Asp 20 25 30

Thr Wing Wing Wing Wing Tyr Arg Asp Wing Wing Read Glu Be Pro Glu His Cys 35 40 45

Be Pro His His Thr Wing Read Arg Gln Wing Lie Leu Cys Trp Gly Asp 50 55 60Leu Met Thr Read Wing Ala Thr Trp Go Gly Thr Asn Leu Glu Asp Gly Gly 65 70 75 80

Lys Gly Gly Be Arg Asp Leu Will Go Tyr Go Asn Thr Asn Go 85 90 95

Gly Leu Lys Phe Arg Gln Leu Leu Trp Phe His Lie Ser Cys Leu Thr 100 105 110

Phe Gly Arg Glu Thr Going To Read Glu Tyr Leu Going To Be Phe Gly Going Trp 115 120 125

lie Arg Thr Pro Pro Wing Tyr Arg Pro Asn Wing Pro Lie Leu Ser 130 135 140

Thr Leu Pro Glu Thr Thr Go Go Arg Arg Arg Asp Arg Gly Arg Ser 145 150 155 160

Pro Arg Arg Arg Thr Pro Be Pro Arg Arg Arg Arg Be Gln Ser Pro 165 170 175

Arg Arg Arg Arg Be Gln Be Arg Glu Be Gln Cys 180 185

<210> 22

<211> 70

<212> PRT

<213> Felis domesticus

<400> 22

Glu lie Cys Pro Wing Go Lys Arg Asp Go Asp Leu Phe Leu Thr Gly 1 5 10 15

Thr Pro Asp Glu Tyr Go Glu Gln Go Wing Gln Tyr Lys Wing Leu Pro 20 25 30

Go Go Leu Glu Asn Wing Arg lie Leu Lys Asn Cys Go Asp Wing Lys 35 40 45

Met Thr Glu Glu Asp Lys Glu Asn Wing Read Leu Read Leu Asp Lys lie 50 55 60

Tyr Thr Ser Pro Read Cys 65 70

<210> 23 <211> 92 <212> PRT <213> Felis domesticus

<400> 23

Go Lys Met Wing Glu Thr Cys Pro lie Phe Tyr Asp Go Phe Phe Ala 15 10 15

Go Wing Asn Gly Asn Glu Leu Leu Leu Asp Leu Being Leu Thr Lys Going 20 25 30

Asn Wing Thr Thru Glu Pro Wing Thr Thr Met Lys Lys lie Gln Asp Cys 35 40 45

Tyr Go Glu Asn Gly Leu Lie Be Arg Go Read Asp Gly Leu Go Met 50 55 60

Thr Thr lie Ser Ser Ser Lys Asp Cys Met Gly Glu Ala Go Gln Asn 65 70 75 80

Thr Go Glu Asp Leu Lys Leu Asn Thr Leu Gly Arg 85 90

<210> 24

<211> 162

<212> PRT

<213> Artificial sequence <220>

<223> Fusion of current 2+ current 1

<400> 24

Go Lys Met Wing Glu Thr Cys Pro lie Phe Tyr Asp Go Phe Phe Ala 15 10 15

Go Wing Asn Gly Asn Glu Leu Leu Leu Asp Leu Be Leu Thr Lys Go 20 25 30

Asn Wing Thr Thru Glu Pro Wing Thr Thr Met Lys Lys lie Gln Asp Cys 35 40 45

Tyr Go Glu Asn Gly Leu Lie Be Arg Go Read Asp Gly Leu Go Met 50 55 60

Thr Thr lie Ser Ser Ser Lys Asp Cys Met Gly Glu Ala Go Gln Asn 65 70 75 80

Thr Go Glu Asp Leu Lys Leu Asn Thr Leu Gly Arg Glu lie Cys Pro 85 90 95

Wing Go Lys Arg Asp Go Asp Leu Phe Leu Thr Gly Thr Pro Asp Glu100 105 110

Tyr Go Glu Gln Go Wing Gln Tyr Lys Wing Leu Pro Go Go Leu Glu 115 120 125

Asn Wing Arg lie Leu Lys Asn Cys Goes Asp Wing Lys Met Thr Glu Glu 130 135 140

Asp Lys Glu Asn Wing Read Serve Read Leu Asp Lys lie Tyr Thr Ser Pro 145 150 155 160

Read Cys

<210> 25 <211> 90 <212> PRT

<213> Felis domesticus <400> 25

Go Lys Met Wing Glu Thr Cys Pro lie Phe Tyr Asp Go Phe Phe Ala 15 10 15

Go Wing Asn Gly Asn Glu Leu Leu Leu Asp Leu Being Leu Thr Lys Going 20 25 30

Asn Wing Thr Thru Glu Pro Wing Thr Thr Met Lys Lys lie Gln Asp Cys 35 40 45

Tyr Go Glu Asn Gly Leu Lie Be Arg Go Read Asp Gly Leu Go Met 50 55 60

lie Ala lie Asn Glu Tyr Cys Met Gly Glu Ala Go Gln Asn Thr Go 65 70 75 80

Glu Asp Leu Lys Leu Asn Thr Leu Gly Arg 85 90

<210> 26

<211> 78

<212> PRT

<213> Felis domesticus

<400> 26

Go Lys Met Wing Glu Thr Cys Pro lie Phe Tyr Asp Go Phe Phe Ala 15 10 15

Go Asn Wing Gly Asn Glu Leu Read Leu Asp Leu Be Read Leu Thr Lys Goes 20 25 30Asn Ala Thr Glu Pro Glu Arg Thr Ala Met Lys Lys lie Gln Asp Cys 35 40 45

Tyr Go Glu Asn Gly Leu Lie Be Arg Go Read Asp Gly Leu Go Met 50 55 60

Pro Be Thr Asn lie Trp Wing Go Lys Gln Phe Arg Thr Pro 65 70 75

<210> 27

<211> 27

<212> PRT

<213> Felis domesticus

<400> 27

Lys Arg Asp Go Asp Leu Phe Leu Thr Gly Thr Pro Asp Glu Tyr Go 1 5 10 15

Glu Gln Go Wing Gln Tyr Lys Wing Leu Pro Go 20 25

<210> 28 <211> 27 <212> PRT

<213> Felis domesticus <400> 28

Lys Ala Leu Pro Go Go Leu Glu Asn Ala Arg lie Leu Lys Asn Cys 15 10 15

Go Asp Wing Lys Met Thr Glu Glu Asp Lys Glu 20 25

<210> 29

<211> 26

<212> PRT

<213> Felis domesticus

<400> 29

Phe Phe Wing Go Wing Asn Gly Asn Glu Leu Leu Leu Asp Leu Ser Leu 15 10 15

Thr Lys Go Asn Wing Thr Glu Pro Glu Arg 20 25

<210> 30

<211> 19

<212> PRT

<213> Felis domesticus <400> 30

Glu Glu Asp Lys Glu Asn Wing Read Leu Be Read Leu Asp Lys lie Tyr Thr 15 10 15

Be Pro Leu

<210> 31

<211> 19

<212> PRT

<213> Felis domesticus

<400> 31

Met Gly Glu Wing Go Gln Asn Thr Go Glu Asp Leu Lys Leu Asn Thr 15 10 15

Read Gly Arg

<210> 32

<211> 16

<212> PRT

<213> Felis domesticus

<400> 32

Glu Glu Asp Lys Glu Asn Wing Read Leu Be Read Leu Asp Lys lie Tyr Thr 15 10 15

<210> 33

<211> 174

<212> PRT

<213> Artificial sequence

<220>

<223> merge with his tag and GGC

<400> 33

Met Go Lys Met Wing Glu Thr Cys Pro lie Phe Tyr Asp Go Phe Phe 1 5 10 15

Wing Go Wing Wing Asn Gly Asn Glu Leu Leu Leu Asp Leu Ser Leu Thr Lys 20 25 30

Go Asn Wing Thr Glu Pro Wing Glu Arg Wing Thr Met Lys Lys lie Gln Asp 35 40 45

Cys Tyr Go Glu Asn Gly Leu Lie Be Arg Go Read Asp Gly Leu Go 50 55 60Met Thr Thr Lie Be Being Lys Asp Cys Met Gly Glu Ala Go Gln 65 70 75 80

Asn Thr Go Glu Asp Leu Lys Leu Asn Thr Leu Gly Arg Glu lie Cys 85 90 95

Pro Wing Go Lys Arg Asp Go Asp Leu Phe Leu Thr Gly Thr Pro Asp 100 105 110

Glu Tyr Go Glu Gln Go Wing Gln Tyr Lys Wing Leu Pro Go Go Leu 115 120 125

Glu Asn Wing Arg lie Leu Lys Asn Cys Going Asp Wing Lys Met Thr Glu 130 135 140

Glu Asp Lys Glu Asn Wing Read Leu Read Leu Asp Lys lie Tyr Thr 145 145 155 155 160

Pro Leu Cys Leu Glu His His His His Gly Gly Cys

170

165

<210> 34

<211> 85

<212> DNA

<213> Artificial sequence

<220>

<223> 1F initiator

<400> 34

gtacatatgg aaatctgccc ggctgttaaa cgtgacçttg acctgttcct gaccggtacc ccggacgaat acgttgaaca ggttg

<210> 35

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<223> 2R primer

<400> 35

ggcagagctt tgtactgagc aacctgttca acgtattc

<210> 36

<211> 75

<212> DNA

<213> Artificial sequence

<220>

<223> 3F primer <400> 36

gctcagtaca aagctctgcc ggttgttctg gaaaacgctc çrtatcctgaa aaactgcgtt gacgctaaaa tgacc

<210> 37

<211> 89

<212> DNA

<213> Artificial sequence

<220>

<223> primer4R

<400> 37

cctctcgagg cacagcgggg aggtgtagat tttgtccagc agggacagag cçttttcttt gtcttcttcg gtcattttag cgtcaacgc

<210> 38

<211> 76

<212> DNA

<213> Artificial sequence

<220>

<223> 5F primer

<400> 38

gtacatatgg ttaaaatggc tgaaacctgc ccgatcttct acgacgtttt cttcgctgtt gctaacggta acgaac

<210> 39

<211> 75

<212> DNA

<213> Artificial sequence

<220>

<223> 6R primer

<400> 39

ggtacgttcc ggttcggtag cgttaacttt ggtcagggac aggtccagca gcagttcgtt accgttagca acagc

40 80 DNA

Artificial sequence

<220>

<223> 7F primer

<400> 40

ctaccgaacc ggaacgtacc gctatgaaaa aaatccagça ctgctacgtt gaaaacggtc tgatctcccg tgttctggac

<210>

<211>

<212>

<213>

<210> 41 <211> 71

<212> DNA

<2i3> Artificial sequence

<220>

<223> 8R initiator

<400> 41

gcttcaccca tgcagtcttt ggaggaggag atggtggtca taaccagacc gtccagaaca cgggagatca g

<210> 42

<211> 75

<212> DNA

<213> Artificial sequence

<220>

<223> 9F primer

<400> 42

caaagactgc atgggtgaag ctgttcagaa caccgttgaa gacctgaaac tgaacaccct gggtcgctcg agagg

<210> 43

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> 10R primer

<400> 43

cctctcgagc gacccagggt g ??? 21

<210> 44

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> binder primer

<400> 44

cgtttaacag ccgggcagat ttcacgaccc agggtgttca gtttc 45

<210> 45

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> primer 12-1

<400> 45

gtacatatgg aaatctgccc ggctgtta 28 <213> Artificial sequence

<220>

<223> primer2-10aa

<400> 51

ggtggaggag gtagcggtgg aggaggtagc gttaaaatgg ctgaaacctg cccgatctt

<210> 52

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> joker 1-5aa

<400> 52

gctacctcct ccaccgcaca gcggggaggt gtagattttg tc

<210> 53

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<223> primer 2-5aa

<400> 53

ggtggaggag gtagcgttaa aatggctgaa acctgcccga tctt

<210> 54

<211> Í75

<212> PRT

<213> Artificial sequence

<220>

<223> FELDl-100aa

<400> 54

Met Go Lys Met Go Wing Glu Thr Cys Pro lie Phe Tyr Asp Go Phe Phe 15 10 15

Wing Go Wing Wing Asn Gly Asn Glu Leu Leu Leu Asp Leu Ser Leu Thr Lys 20 25 30

Go Asn Wing Thr Glu Pro Wing Glu Arg Wing Thr Met Lys Lys lie Gln Asp 35 40 45

Cys Tyr Go Glu Asn Gly Leu Lie Be Arg Go Leu Asp Gly Leu Go 50 55 60

Met Thr Thr lie Be Ser Be Lys Asp Cys Met Gly Glu Ala Go Gln 65 70 75 80Asn Thr Go Glu Asp Leu Lys Leu Asn Thr Leu Gly Arg Gly Gly Gly 85 90 95

Gly Gly Gly Gly Gly Gly Be Glu lie Cys Pro Wing Go Lys Arg Asp 100 105 110

Go Asp Leu Phe Leu Thr Gly Thr Pro Asp Glu Tyr Go Glu Gln Go 115 120 125

Gln Wing Tyr Lys Wing Leu Pro Go Go Leu Glu Asn Wing Arg lie Leu 130 135 140

Lys Asn Cys Go Asp Wing Lys Met Thr Glu Glu Asp Lys Glu Asn Wing 145 150 155 160

Leu Ser Leu Leu Asp Lys lie Tyr Thr Ser Pro Leu Cys Leu Glu

170 175

165

<210> 55

<211> 180

<212> PRT

<213> Artificial sequence

<220>

<223> FELDl-15aa

<400> 55

Met Go Lys Met Wing Glu Thr Cys Pro lie Phe Tyr Asp Go Phe Phe 1 5 10 15

Wing Go Wing Wing Asn Gly Asn Glu Leu Leu Leu Asp Leu Ser Leu Thr Lys 20 25 30

Go Asn Allah Thr Glu Pro Glu Arg Thr Ala Met Lys Lys lie Gln Asp 35 40 45

Cys Tyr Go Glu Asn Gly Leu Lie Be Arg Go Leu Asp Gly Leu Go 50 55 60

Met Thr Thr lie Ser Ser Ser Lys Asp Cys Met Gly Glu Ala Go Gln 65 70 75 80

Asn Thr Go Glu Asp Read Lys Read Asn Thr Read Gly Arg Gly Gly Gly 85 90 95

Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Cly Pro 100 105 110

Wing Go Lys Arg Asp Go Asp Leu Phe Leu Thr Gly Thr Pro Asp Glu115 120 125

Tyr Go Glu Gln Go Wing Gln Tyr Lys Wing Leu Pro Go Go Leu Glu 130 135 140

Asn Wing Arg lie Leu Lys Asn Cys Go Asp Wing Lys Met Thr Glu Glu 145 150 155 160

Asp Lys Glu Asn Wing Read Ser Leu Read Asp Lys Lie Tyr Thr Ser Pro 165 170 175

Leu Cys Leu Glu 180

<210> 56

<211> 165

<212> PRT

<213> Artificial sequence

<220>

<223> FD12

<400> 56

Met Glu lie Cys Pro Wing 1 5

10 15

Gly Thr Pro Asp Glu Tyr Go Glu Gln Go Wing Gln Tyr Lys Wing Leu 20 25 30

Pro Go Go Leu Glu Asn Wing Arg lie Leu Lys Asn Cys Go Asp Wing 35 40 45

Lys Met Thr Glu Glu Asp Lys Glu Asn Wing Read Be Read Leu Asp Lys 50 55 60

lie Tyr Thr Be Pro Read Cys Go Lys Met Wing Glu Thr Cys Pro lie 65 70 75 80

Phe Tyr Asp Go Phe Phe Ward Go Ward Asn Gly Asn Glu Leu Leu Leu 85 90 95

Asp Read Be Read Thr Lys Go Asn Wing Thr Thru Glu Pro Wing Thr Thr 100 105 110

Met Lys Lys lie Gln Asp Cys Tyr Go Glu Asn Gly Leu lie Ser Arg 115 120 125

Go Read Asp Gly Leu Go Met Thr Thr lie Be Ser Be Lys Asp Cys 130 135 140Met Gly Glu Ala Go Gln Asn Thr Go Glu Asp Leu Lys Leu Asn Thr 145 150 155 160

Read Gly Arg Read Le Glu 165

<210> 57

<211> 180

<212> PRT

<213> Artificial sequence <220>

<223> FD12-15aa

<400> 57

Met Glu lie Cys Pro Wing Go Lys Arg Asp Go Asp Leu Phe Leu Thr 1 5 10 15

Gly Thr Pro Asp Glu Tyr Go Glu Gln Go Wing Gln Tyr Lys Wing Leu 20 25 30

Pro Go Go Leu Glu Asn Wing Arg lie Leu Lys Asn Cys Go Asp Wing 35 40 45

Lys Met Thr Glu Glu Asp Lys Glu Asn Wing Read Be Read Leu Asp Lys 50 55 60

lie Tyr Thr Be Pro Read Cys Gly Gly Gly Gly Be Gly Gly Gly Gly Gly 65 70 75 80

Be Gly Gly Gly Gly Be Going Lys Met Wing Glu Thr Cys Pro lie Phe 85 90 95

Tyr Asp Go Phe Phe Ward Go Ward Asn Gly Asn Glu Leu Leu Leu Asp 100 105 110

Leu Be Leu Thr Lys Go Asn Wing Thr Glu Pro Wing Glu Arg Thr Wing 115 115 125

Lys Lys lie Gln Asp Cys Tyr Go Glu Asn Gly Leu lie Ser Arg Go 130 135 140

Leu Asp Gly Leu Going Met Thr Thr lie Being Ser Being Lys Asp Cys Met 145 150 155 160

Gly Glu Wing Go Gln Asn Thr Go Glu Asp Leu Lys Leu Asn Thr Leu 165 170 175Gly Arg Leu Glu 180

<210> 58

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> gamma connector 1

<400> 58

Cys Gly Asp Lys Thr His Thr Ser Pro 1

<210> 59

<211> 6

<212> PRT

<213> Artificial sequence <220>

<223> N-terminal glycine linker

<400> 59

Gly Cys Gly Gly Gly Gly 1 5

<210> 60

<211> 6

<212> PRT

<213> Artificial sequence <220>

<223> N-terminal serine glycine linker

<400> 60

Cys Gly Gly Gly Gly Ser

1 5

<210> 61

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> gcgsggggs connector

<400> 61

Gly Cys Gly Ser Gly Gly Gly Gly Ser 1 5

<210> 62 <211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> C-terminal gamma connector 1

<400> 62

Asp Lys Thr His Thr Be Pro Pro Cys Gly 15 10

<210> 63

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> C-terminal 3 gamma linker

<400> 63

Pro Lys Pro Be Thr Pro Gly Be Ser Gly Gly Wing Pro Gly Gly 1 5 10 15

Cys gly

<210> 64

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> C-terminal glycine linker

<400> 64

Gly Gly Gly Gly

<210> 65

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> C-terminal serine glycine linker

<400> 65

Being Gly Gly Gly Gly Cys 1 5

<210> 66 <211> 10 <212> PRT <213> Artificial Sequence

<220>

<223> GSGGGGSGCG Binder

<400> 66

Gly Gly Gly Gly Gly Gly Gly Gly Cys Gly 15 10

<210> 67

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> glycine lysine linker

<400> 67

Gly Gly Lys Lys Gly Cys 1 5

<210> 68

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223>. linker 2 glycine lysine

<400> 68

Cys Gly Lys

1 5

<210> 69

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> CGGPKPSTPPGS SGGAP

<400> 69

Cys Gly Gly Pro Lys Pro Be Thr Pro Gly Be Ser Gly Gly Wing 15 10 15

Pro

Claims (30)

A composition comprising: (a) a central particle with at least a first attachment site, wherein said central particle is a virus-like particle (VLP) or a viral particle, (b) at least one antigen with at least a second attachment site, wherein said at least one antigen is a Fel d1 protein or a fragment of Fel d1 and wherein (a) and (b) are covalently linked via said at least one first attachment site and said at least one second attachment site. The composition of claim 1, wherein said Fel d1 protein comprises Fel d1 chain 1 and Fel d1 chain 2, wherein said Fel d1 chain 1 is associated with Fel d1 chain 2 for at least a covalent bond. The composition of claim 1 and 2, wherein said Fel d1 protein is a fusion protein comprising Fel d1 chain 1 and Fel d1 chain 2, wherein said Fel d1 chain 1 and Fel d1 chain 2 is a fusion protein. Fel d1 are fused directly or by means of a typical bond or by a spacer, which connects the N-terminus of one chain to the C-terminus of the other chain. The composition of claim 3, wherein said Fel d1 strand 2 is fused by its C-terminus to the N-terminus of said Fel d1 strand 1, either directly or by means of a spacer. The composition of claim 3, wherein said Fel d1 strand 1 is fused by its C-terminus to the N-terminus of said Fel d1 strand 2, either directly or by means of a spacer. A composition according to any one of claims 3-5, wherein said spacer is an amino acid spacer having 1-20 amino acid residues. A composition according to any one of claims 3-6, wherein said fusion protein comprises a sequence of α-mino acids selected from the group consisting of: (a) SEQ ID NO: 24; (C) SEQ ID NO: 55 (d) SEQ ID NO: 56; and (e) SEQ ID NO: 57. A composition according to any one of claims 3-6, wherein said Fel d 1 chain 1 comprises a sequence of SEQ ID NO: 22 or a homologous sequence thereof, wherein said homologous sequence has an identification in The ratio to SEQ ID NO: 22 is greater than 80%, preferably greater than 90% or even more preferably greater than 95%. A composition according to any one of claims 3-6, wherein said Fel d 1 chain 2 comprises a sequence of SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 26, or a homologous sequence. thereof, wherein said homologous sequence has an identification with respect to SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 26 greater than 80%, preferably greater than 90% and even more preferably greater than 95%. . A composition according to any preceding claim, wherein said central particle is a viral particle, and wherein preferably said viral particle is a viral particle of a bacteriophage RNA. A composition according to any one of claims 1-9, wherein said central particle is a virus-like particle, and wherein preferably said virus-like particle is a virus-like particle of a bacteriophage RNA. The composition of claim 10 or 11, wherein said bacteriophage RNA is selected from Qp, fr, GA or AP205. A composition according to any preceding claim wherein said first attachment site comprises or preferably is an amino group, preferably an amino group of a lysine. A composition according to any preceding claim, wherein said second attachment site comprises or preferably is a sulfhydryl group, preferably a sulfhydryl group of a cysteine. A composition according to any preceding claim further comprising a binder. The composition of claim 15, wherein said linker is fused to the C-terminus of said Fel d1 protein or to the C-terminus of said Fel d1 fragment. A vaccine comprising the composition as defined in any one of claims 1-16. A vaccine according to claim 17 further comprising at least one adjuvant. A pharmaceutical composition comprising: (a) the composition according to any one of claims 1-16; and (b) an acceptable pharmaceutical carrier. A process for producing the composition as defined in any one of claims 1-16 comprising: (a) providing a central particle with at least one first attachment site, wherein said central particle is a virus-like particle ( (B) providing at least one antigen with at least a second attachment site, wherein said antigen is a Fel d1 protein or a Fel d1 fragment; and (c) binding said central particle and said at least one antigen to produce said composition, wherein said central particle and said at least one antigen are bound by at least said first and by, at least said second anchorage. Use of the composition as defined in any one of claims 1-16 for the manufacture of a medicament for treating cat allergy. A cat allergy treatment method comprising administering the composition of any one of claims 1-16, or the vaccine as defined in any one of claims 17-18 to a human or non-human mammal, wherein preferably Said non-human mammal is a dog or a cat. The composition of any one of claims 1-16 or the vaccine of any one of claims 17-18 for use in a method for treating cat allergy comprising administering said composition or said vaccine to a human or for a nonhuman mammal, wherein preferably said nonhuman mammal is a dog or a cat. 24. Fel d1 fusion protein comprising Fel d1 chain 1 and Fel d1 chain 2, fused by means of an amino acid spacer, which connects the N-terminus of one chain to the C-terminus of the other chain, wherein said amino acid spacer is an amino acid sequence having 10-30 amino acid residues, and wherein said fusion protein is not a fusion protein comprising chain 1 of SEQ ID 20 NO: 22 fused by (GGGGS) 3 to N-terminal strand 2 of SEQ ID NO: 23, 25 or 26 and wherein said renounced fusion protein is expressed in baculovirus expression system. The Fel d1 fusion protein according to claim 24, wherein said spacer is an amino acid sequence with 2, 3 or 4 GGGGS repeats. The Fel d1 fusion protein according to claim 24 or 25, wherein said Fel d1 fusion protein is produced in E. coli. Fel d1 fusion protein according to any one of claims 24-26, wherein the Fel d1 chain 2 is located at the N-terminus of the fusion protein. Fel d1 fusion protein according to any one of claims 24-26, wherein the Fel d1 chain 1 is located at the N-terminus of the fusion protein. A Fel d1 fusion protein according to any one of claims 24-28 comprising an amino acid sequence selected from the group consisting of: (a) SEQ ID NO: 54 (b) SEQ ID NO: 55; and (c) SEQ ID NO: 57. 30. Fel d1 fusion protein comprising an amino acid sequence of SEQ ID NO: 57, wherein said fusion protein is produced in E. coli.